bluuebunny/crossref_metadata_2025 huggingface dataset:
Postnatal mammary gland development requires macrophages and eosinophils
Authors: Gouon-Evans Valérie , Rothenberg Marc E. , Pollard Jeffrey W.
Published: April 2021

Interactions between mammary epithelial and mesenchymal cells including fibroblasts and adipocytes are crucial for the proper postnatal development of the mammary ductal tree. Often overlooked, however, are the migrant cells that enter tissues at different stages of development. In this paper we identify two such cell types, macrophages and eosinophils, that are recruited around the growing terminal end buds (TEBs) during postnatal development. An important role for leukocytes in mammary gland ductal outgrowth is first demonstrated by depleting mice of leukocytes using sub-lethal γ-irradiation. This treatment results in a curtailment of mammary gland epithelial development that is completely rescued by bone-marrow transplantation, concurrent with a restoration of macrophage and eosinophil recruitment around the growing ducts. Using mice homozygous for a null mutation in the gene for CSF1 (Csfmop/Csfmop), the major growth factor for macrophages, we show that in the absence of CSF1, the population of macrophages in mammary glands is depleted. In this mutant, the formation of TEBs, their outgrowth into the fat pad and the branching of the resultant ducts are all impaired. Similarly, by using mice homozygous for a null mutation in the gene for eotaxin, a major chemokine for local recruitment of eosinophils in tissue, we identify eotaxin as the necessary and sufficient chemokine responsible for eosinophil recruitment around TEBs. In the absence of eosinophils, mammary gland branch formation and to a lesser extent TEB formation are reduced. Our data show that CSF1-regulated macrophages, in collaboration with eotaxin-regulated eosinophils, have essential and complementary functions in regulating the branching morphogenesis of the mammary gland.
Hex is a transcriptional repressor that contributes to anterior identity and suppresses Spemann organiser function
Authors: Brickman Joshua M. , Jones C. Michael , Clements Melanie , Smith J. C. , Beddington Rosa S. P.
Published: April 2021

One of the earliest markers of anterior asymmetry in vertebrate embryos is the transcription factor Hex. We find that Hex is a transcriptional repressor that can be converted to an activator by fusing full length Hex to two copies of the minimal transcriptional activation domain of VP16 together with the flexible hinge region of the λ repressor (Hex-λVP2). Retention of the entire Hex open reading frame allows one to examine Hex function without disrupting potential protein-protein interactions. Expression of Hex-λVP2 in Xenopus inhibits expression of the anterior marker Cerberus and results in anterior truncations. Such embryos have multiple notochords and disorganised muscle tissue. These effects can occur in a cell non-autonomous manner, suggesting that one role of wild-type Hex is to specify anterior structures by suppressing signals that promote dorsal mesoderm formation. In support of this idea, over-expression of wild-type Hex causes cell non-autonomous dorso-anteriorisation, as well as cell autonomous suppression of dorsal mesoderm. Suppression of dorsal mesoderm by Hex is accompanied by the down-regulation of Goosecoid and Chordin, while induction of dorsal mesoderm by Hex-λVP2 results in activation of these genes. Transient transfection experiments in ES cells suggest that Goosecoid is a direct target of Hex. Together, our results support a model in which Hex suppresses organiser activity and defines anterior identity.
The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation
Authors: Barbera Juan Pedro Martinez , Clements Melanie , Thomas Paul , Rodriguez Tristan , Meloy Denise , Kioussis Dimitris , Beddington Rosa S. P.
Published: April 2021

The homeobox gene Hex is expressed in the anterior visceral endoderm (AVE) and rostral definitive endoderm of early mouse embryos. Later, Hex transcripts are detected in liver, thyroid and endothelial precursor cells. A null mutation was introduced into the Hex locus by homologous recombination in embryonic stem cells. Hex mutant embryos exhibit varying degrees of anterior truncation as well as liver and thyroid dysplasia. The liver diverticulum is formed but migration of hepatocytes into the septum transversum fails to occur. Development of the thyroid is arrested at the thyroid bud stage at 9.5 dpc. Brain defects are restricted to the rostral forebrain and have a caudal limit at the zona limitans intrathalamica, the boundary between dorsal and ventral thalamus. Analysis of Hex−/− mutants at early stages shows that the prospective forebrain ectoderm is correctly induced and patterned at 7.5 days post coitum (dpc), but subsequently fails to develop. AVE markers are expressed and correctly positioned but development of rostral definitive endoderm is greatly disturbed in Hex−/− embryos. Chimeric embryos composed of Hex−/− cells developing within a wild-type visceral endoderm show forebrain defects indicating that Hex is required in the definitive endoderm. All together, these results demonstrate that Hex function is essential in definitive endoderm for normal development of the forebrain, liver and thyroid gland.
Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation
Authors: Haraguchi R. , Suzuki K. , Murakami R. , Sakai M. , Kamikawa M. , Kengaku M. , Sekine K. , Kawano H. , Kato S. , Ueno N. , Yamada G.
Published: April 2021

The molecular mechanisms underlying the development of the external genitalia in mammals have been very little examined. Recent gene knockout studies have suggested that the developmental processes of its anlage, the genital tubercle (GT), have much in common with those of limb buds. The Fgf genes have been postulated as regulating several downstream genes during organogenesis. Fgf8 was expressed in the distal urethral plate epithelium of the genital tubercle (GT) together with other markers such as the Msx1, Fgf10, Hoxd13 and Bmp4 expressed in the mesenchyme. To analyze the role of the FGF system during GT formation, an in vitro organ culture system was utilized. It is suggested that the distal urethral plate epithelium of GT, the Fgf8-expressing region, regulates the outgrowth of GT. Ectopic application of FGF8 beads to the murine GT induced mesenchymal gene expression, and also promoted the outgrowth of the GT. Experiments utilizing anti-FGF neutralizing antibody suggested a growth-promoting role for FGF protein(s) in GT outgrowth. In contrast, despite its vital role during limb- bud formation, Fgf10 appears not to be primarily essential for initial outgrowth of GT, as extrapolated from Fgf10−/− GTs. However, the abnormal external genitalia development of Fgf10−/− perinatal mice suggested the importance of Fgf10 in the development of the glans penis and the glans clitoridis. These results suggest that the FGF system is a key element in orchestrating GT development.
The bHLH transcription factor Hand2 plays parallel roles in zebrafish heart and pectoral fin development
Authors: Yelon Deborah , Ticho Baruch , Halpern Marnie E. , Ruvinsky Ilya , Ho Robert K. , Silver Lee M. , Stainier Didier Y. R.
Published: April 2021

The precursors of several organs reside within the lateral plate mesoderm of vertebrate embryos. Here, we demonstrate that the zebrafish hands off locus is essential for the development of two structures derived from the lateral plate mesoderm-the heart and the pectoral fin. hands off mutant embryos have defects in myocardial development from an early stage: they produce a reduced number of myocardial precursors, and the myocardial tissue that does form is improperly patterned and fails to maintain tbx5 expression. A similar array of defects is observed in the differentiation of the pectoral fin mesenchyme: small fin buds form in a delayed fashion, anteroposterior patterning of the fin mesenchyme is absent and tbx5 expression is poorly maintained. Defects in these mesodermal structures are preceded by the aberrant morphogenesis of both the cardiogenic and forelimbforming regions of the lateral plate mesoderm. Molecular analysis of two hands off alleles indicates that the hands off locus encodes the bHLH transcription factor Hand2, which is expressed in the lateral plate mesoderm starting at the completion of gastrulation. Thus, these studies reveal early functions for Hand2 in several cellular processes and highlight a genetic parallel between heart and forelimb development.
Transforming growth factor β3 induces cell death during the first stage of mammary gland involution
Authors: Nguyen Andrew V. , Pollard Jeffrey W.
Published: April 2021

Involution of the mammary gland following weaning is divided into two distinct phases. Initially, milk stasis results in the induction of local factors that cause apoptosis in the alveolar epithelium. Secondly after a prolonged absence of suckling, the consequent decline in circulating lactogenic hormone concentrations initiates remodeling of the mammary gland to the virgin-like state. We have shown that immediately following weaning TGFβ3 mRNA and protein is rapidly induced in the mammary epithelium and that this precedes the onset of apoptosis. Unilateral inhibition of suckling and hormonal reconstitution experiments showed that TGFβ3 induction is regulated by milk stasis and not by the circulating hormonal concentration. Directed expression of TGFβ3 in the alveolar epithelium of lactating mice using a β-lactoglobulin promoter mobilized SMAD4 translocation to the nucleus and caused apoptosis of these cells, but not tissue remodeling. Transplantation of neonatal mammary tissue derived from TGFβ3 null mutant mice into syngenic hosts resulted in a significant inhibition of cell death compared to wild-type mice upon milk stasis. These results provide direct evidence that TGFβ3 is a local mammary factor induced by milk stasis that causes apoptosis in the mammary gland epithelium during involution.
Structural requirements for Notch signalling with Delta and Serrate during the development and patterning of the wing disc of Drosophila
Authors: Lawrence Nicola , Klein Thomas , Brennan Keith , Arias Alfonso Martinez
Published: April 2021

The Delta and Serrate proteins interact with the extracellular domain of the Notch receptor and initiate signalling through the receptor. The two ligands are very similar in structure and have been shown to be interchangeable experimentally; however, loss of function analysis indicates that they have different functions during development and analysis of their signalling during wing development indicates that the Fringe protein can discriminate between the two ligands. This raises the possibility that the signalling of Delta and Serrate through Notch requires different domains of the Notch protein. Here we have tested this possibility by examining the ability of Delta and Serrate to interact and signal with Notch molecules in which different domains had been deleted. This analysis has shown that EGF-like repeats 11 and 12, the RAM-23 and cdc10/ankyrin repeats and the region C-terminal to the cdc10/ankyrin repeats of Notch are necessary for both Delta and Serrate to signal via Notch. They also indicate, however, that Delta and Serrate utilise EGF-like repeats 24-26 of Notch for signalling, but there are significant differences in the way they utilise these repeats.
Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo
Authors: Ashe Hilary L. , Mannervik Mattias , Levine Michael
Published: April 2021

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFβ signaling molecules, Decapentaplegic (Dpp) and Screw (Scw). Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation (Sog) and a newly identified transcriptional repressor, Brinker (Brk), which are expressed in neurogenic regions that abut the dorsal ectoderm. Here we examine the expression of a number of Dpp target genes in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. We also present evidence that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase.
The homeodomain protein CePHOX2/CEH-17 controls antero-posterior axonal growth in C. elegans
Authors: Pujol Nathalie , Torregrossa Pascal , Ewbank Jonathan J. , Brunet Jean-François
Published: April 2021

An essential aspect of a neuron’s identity is the pattern of its axonal projections. In C. elegans, axons extend either longitudinally or circumferentially in response to distinct molecular cues, some of which have been identified. It is currently unclear, however, how the differential capacity to respond to these cues is transcriptionally implemented in distinct neuronal subtypes. Here, we characterise a C. elegans paired-like homeobox gene, CePhox2/ceh-17, expressed in five head neurons, ALA and the 4 SIAs, all of which project axons towards the tail along the lateral and sublateral cords. Abrogation of ceh-17 function, while leaving intact many phenotypic traits of these neurons, disrupts their antero-posterior axonal elongation beyond the mid-body region. Conversely, ectopic expression of ceh-17 in the mechanoreceptors, several of which are known to pioneer their tract, leads to exaggerated longitudinal axonal outgrowth. Thus, ceh-17 is a novel gene involved in fasciculation-independent longitudinal axonal navigation.
Deletion of a silencer element disruptsH19imprinting independently of a DNA methylation epigenetic switch
Authors: Drewell Robert A. , Brenton James D. , Ainscough Justin F.-X. , Barton Sheila C. , Hilton Kathy J. , Arney Katharine L. , Dandolo Luisa , Surani M. Azim
Published: April 2021

The H19 imprinted gene is silenced when paternally inherited and active only when inherited maternally. This is thought to involve a cis-acting control region upstream of H19 that is responsible for regulating a number of functions including DNA methylation, asynchronous replication of parental chromosomes and an insulator. Here we report on the function of a 1.2 kb upstream element in the mouse, which was previously shown to function as a bi-directional silencer in Drosophila. The cre-loxP-mediated targeted deletion of the 1.2 kb region had no effect on the maternal allele. However, there was loss of silencing of the paternal allele in many endodermal and other tissues. The pattern of expression was very similar to the expression pattern conferred by the enhancer elements downstream of H19. We could not detect an effect on the expression of the neighbouring imprinted Igf2 gene, suggesting that the proposed boundary element insulating this gene from the downstream enhancers was unaffected. Despite derepression of the paternal H19 allele, the deletion surprisingly did not affect the differential DNA methylation of the locus, which displayed an appropriate epigenetic switch in the parental germlines. Furthermore, the characteristic asynchronous pattern of DNA replication at H19 was also not disrupted by the deletion, suggesting that the sequences that mediate this were also intact. The silencer is therefore part of a complex cis-regulatory region upstream of the H19 gene and acts specifically to ensure the repression of the paternal allele, without a predominant effect on the epigenetic switch in the germline.
p57Kip2 regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina
Authors: Dyer Michael A. , Cepko Constance L.
Published: April 2021

A precise balance between proliferation and differentiation must be maintained during retinal development to obtain the correct proportion of each of the seven cell types found in the adult tissue. Cyclin kinase inhibitors can regulate cell cycle exit coincident with induction of differentiation programs during development. We have found that the p57Kip2 cyclin kinase inhibitor is upregulated during G1/G0 in a subset of retinal progenitor cells exiting the cell cycle between embryonic day 14.5 and 16.5 of mouse development. Retroviral mediated overexpression of p57Kip2 in embryonic retinal progenitor cells led to premature cell cycle exit. Retinae from mice lacking p57Kip2 exhibited inappropriate S-phase entry and apoptotic nuclei were found in the region where p57Kip2 is normally expressed. Apoptosis precisely compensated for the inappropriate proliferation in the p57Kip2-deficient retinae to preserve the correct proportion of the major retinal cell types. Postnatally, p57Kip2 was found to be expressed in a novel subpopulation of amacrine interneurons. At this stage, p57Kip2 did not regulate proliferation. However, perhaps reflecting its role during this late stage of development, animals lacking p57Kip2 showed an alteration in amacrine subpopulations. p57Kip2 is the first gene to be implicated as a regulator of amacrine subtype/subpopulation development. Consequently, we propose that p57Kip2 has two roles during retinal development, acting first as a cyclin kinase inhibitor in mitotic progenitor cells, and then playing a distinct role in neuronal differentiation.
Zebrafishkitmutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration
Authors: Rawls John F. , Johnson Stephen L.
Published: April 2021

Fin regeneration in adult zebrafish is accompanied by reestablishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.
EGF receptor attenuates Dpp signaling and helps to distinguish the wing and leg cell fates inDrosophila
Authors: Kubota Kazumasa , Goto Satoshi , Eto Kazuhiro , Hayashi Shigeo
Published: April 2021

Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. We report here that the receptor tyrosine kinase EGFR antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and EGFR, we show that nuclear transduction of the two signals peaks at different position along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice.
sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development
Authors: Miller Craig T. , Schilling, Thomas F. , Lee, Kyu-Ho , Parker Jewel , Kimmel Charles B.
Published: April 2021

Mutation of sucker (suc) disrupts development of the lower jaw and other ventral cartilages in pharyngeal segments of the zebrafish head. Our sequencing, cosegregation and rescue results indicate that suc encodes an Endothelin-1 (Et-1). Like mouse and chick Et-1, suc/et-1 is expressed in a central core of arch paraxial mesoderm and in arch epithelia, both surface ectoderm and pharyngeal endoderm, but not in skeletogenic neural crest. Long before chondrogenesis, suc/et-1 mutant embryos have severe defects in ventral arch neural crest expression of dHAND, dlx2, msxE, gsc, dlx3 and EphA3 in the anterior arches. Dorsal expression patterns are unaffected. Later in development, suc/et-1 mutant embryos display defects in mesodermal and endodermal tissues of the pharynx. Ventral premyogenic condensations fail to express myoD, which correlates with a ventral muscle defect. Further, expression of shh in endoderm of the first pharyngeal pouch fails to extend as far laterally as in wild types. We use mosaic analyses to show that suc/et-1 functions nonautonomously in neural crest cells, and is thus required in the environment of postmigratory neural crest cells to specify ventral arch fates. Our mosaic analyses further show that suc/et-1 nonautonomously functions in mesendoderm for ventral arch muscle formation. Collectively our results support a model for dorsoventral patterning of the gnathostome pharyngeal arches in which Et-1 in the environment of the postmigratory cranial neural crest specifies the lower jaw and other ventral arch fates.
Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension
Authors: Kwong Wing Yee , Wild Arthur E. , Roberts Paul , Willis Antony C. , Fleming Tom P.
Published: April 2021

Epidemiological studies have indicated that susceptibility of human adults to hypertension and cardiovascular disease may result from intrauterine growth restriction and low birth weight induced by maternal undernutrition. Although the ‘foetal origins of adult disease’ hypothesis has significant relevance to preventative healthcare, the origin and biological mechanisms of foetal programming are largely unknown. Here, we investigate the origin, embryonic phenotype and potential maternal mechanisms of programming within an established rat model. Maternal low protein diet (LPD) fed during only the preimplantation period of development (0-4.25 days after mating), before return to control diet for the remainder of gestation, induced programming of altered birthweight, postnatal growth rate, hypertension and organ/body-weight ratios in either male or female offspring at up to 12 weeks of age. Preimplantation embryos collected from dams after 0-4.25 days of maternal LPD displayed significantly reduced cell numbers, first within the inner cell mass (ICM; early blastocyst), and later within both ICM and trophectoderm lineages (mid/late blastocyst), apparently induced by a slower rate of cellular proliferation rather than by increased apoptosis. The LPD regimen significantly reduced insulin and essential amino acid levels, and increased glucose levels within maternal serum by day 4 of development. Our data indicate that long-term programming of postnatal growth and physiology can be induced irreversibly during the preimplantation period of development by maternal protein undernutrition. Further, we propose that the mildly hyperglycaemic and amino acid-depleted maternal environment generated by undernutrition may act as an early mechanism of programming and initiate conditions of ‘metabolic stress’, restricting early embryonic proliferation and the generation of appropriately sized stem-cell lineages.
Effect of single and compound knockouts of estrogen receptors α (ERα) and β (ERβ) on mouse reproductive phenotypes
Authors: Dupont Sonia , Krust Andrée , Gansmuller Anne , Dierich Andrée , Chambon Pierre , Mark Manuel
Published: April 2021

The functions of estrogen receptors (ERs) in mouse ovary and genital tracts were investigated by generating null mutants for ERα (ERαKO), ERβ (ERβKO) and both ERs (ERαβKO). All ERαKO females are sterile, whereas ERβKO females are either infertile or exhibit variable degrees of subfertility. Mast cells present in adult ERαKO and ERαβKO ovaries could participate in the generation of hemorrhagic cysts. Folliculogenesis proceeds normally up to the large antral stage in both ERαKO and ERβKO adults, whereas large antral follicles of ERα+/−/ERβKO and ERαβKO adults are markedly deficient in granulosa cells. Similarly, prematurely developed follicles found in prepubertal ERαKO ovaries appear normal, but their ERαβKO counterparts display only few granulosa cell layers. Upon superovulation treatment, all prepubertal ERαKO females form numerous preovulatory follicles of which the vast majority do not ovulate. The same treatment fails to elicit the formation of preovulatory follicles in half of the ERβKO mice and in all ERα+/−/ERβKO mice. These and other results reveal a functional redundancy between ERα and ERβ for ovarian folliculogenesis, and strongly suggest that (1) ERβ plays an important role in mediating the stimulatory effects of estrogens on granulosa cell proliferation, (2) ERα is not required for follicle growth under wild type conditions, while it is indispensable for ovulation, and (3) ERα is also necessary for interstitial glandular cell development. Our data also indicate that ERβ exerts some function in ERαKO uterus and vagina. ERαβKO granulosa cells localized within degenerating follicles transform into cells displaying junctions that are unique to testicular Sertoli cells. From the distribution pattern of anti-Müllerian hormone (AMH) in ERαβKO ovaries, it is unlikely that an elevated AMH level is the cause of Sertoli cell differentiation. Our results also show that cell proliferation in the prostate and urinary bladder of old ERβKO and ERαβKO males is apparently normal.
Independent regulation of Dlx2 expression in the epithelium and mesenchyme of the first branchial arch
Authors: Thomas Bethan L. , Liu Jen Kuei , Rubenstein John L. R. , Sharpe Paul T.
Published: April 2021

Dlx2, a member of the distal-less gene family, is expressed in the first branchial arch, prior to the initiation of tooth development, in distinct, non-overlapping domains in the mesenchyme and the epithelium. In the mesenchyme Dlx2 is expressed proximally, whereas in oral epithelium it is expressed distally. Dlx2 has been shown to be involved in the patterning of the murine dentition, since loss of function of Dlx1 and Dlx2 results in early failure of development of upper molar teeth. We have investigated the regulation of Dlx2 expression to determine how the early epithelial and mesenchymal expression boundaries are maintained, to help to understand the role of these distinct expression domains in patterning of the dentition.

Transgenic mice produced with a lacZ reporter construct, containing 3.8 kb upstream sequence of Dlx2, led to the mapping of regulatory regions driving epithelial but not mesenchymal expression in the first branchial arch. We show that the epithelial expression of Dlx2 is regulated by planar signalling by BMP4, which is coexpressed in distal oral epithelium. Mesenchymal expression is regulated by a different mechanism involving FGF8, which is expressed in the overlying epithelium. FGF8 also inhibits expression of Dlx2 in the epithelium by a signalling pathway that requires the mesenchyme. Thus, the signalling molecules BMP4 and FGF8 provide the mechanism for maintaining the strict epithelial and mesenchymal expression domains of Dlx2 in the first arch.
Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar)
Authors: Reifers Frank , Walsh Emily C. , Léger Sophie , Stainier Didier Y. R. , Brand Michael
Published: April 2021

Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors, but the extracellular signals specifying heart precursor gene expression are not known. We describe here that the secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors, particularly during initiation of cardiac gene expression. fgf8 is mutated in acerebellar (ace) mutants, and homozygous mutant embryos do not establish normal circulation, although vessel formation is only mildly affected. In contrast, heart development, in particular of the ventricle, is severely abnormal in acerebellar mutants. Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells: fgf8 is expressed in cardiac precursors and later in the heart ventricle. Fgf8 is required for the earliest stages of nkx2.5 and gata4, but not gata6, expression in cardiac precursors. Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 RNA, or by implanting a Fgf8-coated bead into the heart primordium. Pharmacological inhibition of Fgf signalling during formation of the heart primordium phenocopies the acerebellar heart phenotype, confirming that Fgf signaling is required independently of earlier functions during gastrulation. These findings show that fgf8/acerebellar is required for induction and patterning of myocardial precursors.
Regulation of neurogenesis by interactions between HEN1 and neuronal LMO proteins
Authors: Bao Jianxin , Talmage David A. , Role Lorna W. , Gautier Jean
Published: April 2021

Basic-helix-loop-helix transcription factors regulate neurogenesis and neuronal differentiation by as yet unknown mechanisms. We show that an embryonic neuronal-specific basic-helix-loop-helix protein, HEN1 (also known as NSCL1 or NHLH), interacts with ‘LIM only’ proteins. Examination of the expression patterns of XHEN1 and XLMO-3, the Xenopus homologues of these human genes, reveals extensive overlap during early neurogenesis: at the onset of gastrulation on the dorsal side of the blastopore lip and, subsequently, in the prospective neural plate. Binding of XLMO-3 increases the transcriptional activity of XHEN1 in vivo. Co-expression of these two genes in Xenopus embryos induces a cascade of expression of neuronal-specific basic-helix-loop-helix proteins that leads to neuronal differentiation. We propose that XHEN1, in concert with XLMO-3, is a critical regulator of neurogenesis.
Hhex and Scl function in parallel to regulate early endothelial and blood differentiation in zebrafish
Authors: Liao Wayne , Ho Chi-Yip , Yan Yi Lin , Postlethwait John , Stainier Didier Y. R.
Published: April 2021

During embryogenesis, endothelial and blood precursors are hypothesized to arise from a common progenitor, the hemangioblast. Several genes that affect the differentiation of, or are expressed early in, both the endothelial and blood lineages may in fact function at the level of the hemangioblast. For example, the zebrafish cloche mutation disrupts the differentiation of both endothelial and blood cells. The transcription factor gene scl is expressed in both endothelial and blood lineages from an early stage and can regulate their differentiation. Here we report that in zebrafish the homeobox gene hhex (previously called hex) is also expressed in endothelial and blood lineages from an early stage. We find that hhex expression in these lineages is significantly reduced in cloche mutant embryos, indicating that hhex functions downstream of cloche to regulate endothelial and blood differentiation. Ectopic expression of hhex through injection of a DNA construct leads to the premature and ectopic expression of early endothelial and blood differentiation genes such as fli1, flk1 and gata1, indicating that Hhex can positively regulate endothelial and blood differentiation. However, analysis of a hhex deficiency allele shows that hhex is not essential for early endothelial and blood differentiation, suggesting that another gene, perhaps scl, compensates for the absence of Hhex function. Furthermore, we find that hhex and scl can induce each other’s expression, suggesting that these two genes cross-regulate each other during early endothelial and blood differentiation. Together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation.
Hes1is a negative regulator of inner ear hair cell differentiation
Authors: Zheng J. Lisa , Shou Jianyong , Guillemot Francois , Kageyama Ryoichiro , Gao Wei-Qiang
Published: April 2021

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, we have focused on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. We report here that targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions which are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells.
Repressive and restrictive mesodermal interactions with gut endoderm: possible relation to Meckel’s Diverticulum
Authors: Bossard Pascale , Zaret Kenneth S.
Published: April 2021

The midgut and hindgut endoderm of the mouse embryo give rise to the intestinal epithelium, yet it is not known how the intestinal program is chosen in contrast to other endoderm-derived cell types. Previous tissue explant studies with embryos at 8.5 to 11.5 days gestation (d) showed that when the gut mesoderm is removed from the prospective intestinal endoderm, the endoderm activates the expression of liver-specific genes such as serum albumin, demonstrating the endoderm’s pluripotence. This reversible repression of liver genes does not affect the expression of the endodermal transcription factors HNF3 and GATA4, nor these factors’ ability to engage target sites in chromatin. We have now found that at 13.5 d, the mesoderm gains a second inhibitory activity, resulting in the irreversible loss of expression of HNF3 (Foxa2) and GATA factors in the endoderm and the absence of factors binding to their target sites in chromatin. The second inhibitory activity causes the endoderm to lose the potential to activate a liver gene, and this restriction precedes the normal cytodifferentiation of the intestinal epithelium. In summary, two inhibitory interactions with mesoderm successively restrict the developmental potential of the gut endoderm, leading to intestinal differentiation. We also observed rare gut bud structures in midgestation embryos that appear to represent murine examples of Meckel’s Diverticulum, a congenital abnormality in human development. The absence of restrictive mesodermal interactions could explain how Meckel’s diverticula express diverse non-intestinal, endoderm-derived cell types.
An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis
Authors: Moerlooze Laurence De , Spencer-Dene Bradley , Revest Jean-Michel , Hajihosseini Mohammad , Rosewell Ian , Dickson Clive
Published: April 2021

The fibroblast growth factor receptor 2 gene is differentially spliced to encode two transmembrane tyrosine kinase receptor proteins that have different ligandbinding specificities and exclusive tissue distributions. We have used Cre-mediated excision to generate mice lacking the IIIb form of fibroblast growth factor receptor 2 whilst retaining expression of the IIIc form. Fibroblast growth factor receptor 2(IIIb) null mice are viable until birth, but have severe defects of the limbs, lung and anterior pituitary gland. The development of these structures appears to initiate, but then fails with the tissues undergoing extensive apoptosis. There are also developmental abnormalities of the salivary glands, inner ear, teeth and skin, as well as minor defects in skull formation. Our findings point to a key role for fibroblast growth factor receptor 2(IIIb) in mesenchymal-epithelial signalling during early organogenesis.
Sperm entry induces polarity in fucoid zygotes
Authors: Hable Whitney E. , Kropf Darryl L.
Published: April 2021

Fucoid zygotes establish a rhizoid-thallus growth axis in response to environmental signals; however, these extrinsic cues are not necessary for polarization, suggesting that zygotes may have inherent polarity. The hypothesis that sperm entry provides a default pathway for polarization of zygotes cultured in the absence of environmental signals was tested, and was supported by several lines of evidence. First, an F-actin patch, a cortical marker of the rhizoid pole, formed at the sperm entry site within minutes of fertilization. Second, the sperm entry site predicted the site of polar adhesive secretion (the first morphological manifestation of the rhizoid pole) and the position of rhizoid outgrowth. Third, when fertilization was restricted to one hemisphere of the egg, rhizoid outgrowth always occurred from that hemisphere. Fourth, delivery of sperm to one location within a population of eggs resulted in polarization of both adhesive secretion and rhizoid outgrowth toward the sperm source. Finally, induction of polyspermy using low sodium seawater increased the frequency of formation of two rhizoids. Sperm entry therefore provides an immediate default axis that can later be overridden by environmental cues.
Requirements for thekitreceptor tyrosine kinase during regeneration of zebrafish fin melanocytes
Authors: Rawls John F. , Johnson Stephen L.
Published: April 2021

Embryonic neural crest-derived melanocytes and their precursors express the kit receptor tyrosine kinase and require its function for their migration and survival. However, mutations in kit also cause deficits in melanocytes that make up adult pigment patterns, including melanocytes that re-establish the zebrafish fin stripes during regeneration. As adult melanocytes in mice and zebrafish are generated and maintained by stem cell populations that are presumably established during embryonic development, it has been proposed that adult phenotypes in kit mutants result from embryonic requirements for kit. We have used a temperature-sensitive zebrafish kit mutation to show that kit is required during adult fin regeneration to promote melanocyte differentiation, rather than during embryonic stages to establish their stem cell precursors. We also demonstrate a transient role for kit in promoting the survival of newly differentiated regeneration melanocytes.
TheDrosophilatumor suppressor genelethal(2)giant larvaeis required for the emission of the Decapentaplegic signal
Authors: Arquier Nathalie , Perrin Laurent , Manfruelli Pascal , Sémériva Michel
Published: April 2021

The Drosophila tumor suppressor gene lethal(2) giant larvae (lgl) encodes a cytoskeletal protein required for the change in shape and polarity acquisition of epithelial cells, and also for asymmetric division of neuroblasts. We show here that lgl participates in the emission of Decapentaplegic (Dpp), a member of the transforming growth factor β (TGFβ) family, in various developmental processes.

During embryogenesis, lgl is required for the dpp-dependent transcriptional activation of zipper (zip), which encodes the non-muscle myosin heavy chain (NMHC), in the dorsalmost ectodermal cells – the leading edge cells. The embryonic expression of known targets of the dpp signaling pathway, such as labial or tinman was abolished or strongly reduced in lgl mutants. lgl mutant cuticles exhibited phenotypes resembling those observed in mutated partners of the dpp signaling pathway. In addition, lgl was required downstream of dpp and upstream of its receptor Thickveins (Tkv) for the dorsoventral patterning of the ectoderm. During larval development, the expression of spalt, a dpp target, was abolished in mutant wing discs, while it was restored by a constitutively activated form of Tkv (TkvQ253D). Taking into account that the activation of dpp expression was unaffected in the mutant, this suggests that lgl function is not required downstream of the Dpp receptor. Finally, the function of lgl responsible for the activation of Spalt expression appeared to be required only in the cells that produce Dpp, and lgl mutant somatic clones behaved non autonomously. We therefore position the activity of lgl in the cells that produce Dpp, and not in those that respond to the Dpp signal. These results are consistent with a same role for lgl in exocytosis and secretion as that proposed for its yeast ortholog sro7/77 and lgl might function in parallel or independently of its well-documented role in the control of epithelial cell polarity.
Hoxd10induction and regionalization in the developing lumbosacral spinal cord
Authors: Lance-Jones Cynthia , Omelchenko Natalia , Bailis Anya , Lynch Stephen , Sharma Kamal
Published: April 2021

We have used Hoxd10 expression as a primary marker of the lumbosacral region to examine the early programming of regional characteristics within the posterior spinal cord of the chick embryo. Hoxd10 is uniquely expressed at a high level in the lumbosacral cord, from the earliest stages of motor column formation through stages of motoneuron axon outgrowth. To define the time period when this gene pattern is determined, we assessed Hoxd10 expression after transposition of lumbosacral and thoracic segments at early neural tube stages. We present evidence that there is an early prepattern for Hoxd10 expression in the lumbosacral neural tube; a prepattern that is established at or before stages of neural tube closure. Cells within more posterior lumbosacral segments have a greater ability to develop high level Hoxd10 expression than the most anterior lumbosacral segments or thoracic segments. During subsequent neural tube stages, this prepattern is amplified and stabilized by environmental signals such that all lumbosacral segments acquire the ability to develop high levels of Hoxd10, independent of their axial environment. Results from experiments in which posterior neural segments and/or paraxial mesoderm segments were placed at different axial levels suggest that signals setting Hoxd10 expression form a decreasing posterior-to-anterior gradient. Our experiments do not, however, implicate adjacent paraxial mesoderm as the only source of graded signals. We suggest, instead, that signals from more posterior embryonic regions influence Hoxd10 expression after the early establishment of a regional prepattern. Concurrent analyses of patterns of LIM proteins and motor column organization after experimental surgeries suggest that the programming of these characteristics follows similar rules.
The BMP signaling pathway is required together with the FGF pathway for notochord induction in the ascidian embryo
Authors: Darras Sébastien , Nishida Hiroki
Published: April 2021

The 40 notochord cells of the ascidian tadpole invariably arise from two different lineages: the primary (A-line) and the secondary (B-line) lineages. It has been shown that the primary notochord cells are induced by presumptive endoderm blastomeres between the 24-cell and the 64-cell stage. Signaling through the fibroblast growth factor (FGF) pathway is required for this induction. We have investigated the role of the bone morphogenetic protein (BMP) pathway in ascidian notochord formation. HrBMPb (the ascidian BMP2/4 homologue) is expressed in the anterior endoderm at the 44-cell stage before the completion of notochord induction. The BMP antagonist Hrchordin is expressed in a complementary manner in all surrounding blastomeres and appears to be a positive target of the BMP pathway. Unexpectedly, chordin overexpression reduced formation of both primary and secondary notochord. Conversely, primary notochord precursors isolated prior to induction formed notochord in presence of BMP-4 protein. While bFGF protein had a similar activity, notochord precursors showed a different time window of competence to respond to BMP-4 and bFGF. Our data are consistent with bFGF acting from the 24-cell stage, while BMP-4 acts during the 44-cell stage. However, active FGF signaling was also required for induction by BMP-4. In the secondary lineage, notochord specification also required two inducing signals: an FGF signal from anterior and posterior endoderm from the 24-cell stage and a BMP signal from anterior endoderm during the 44-cell stage.
A HOX complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer
Authors: Marty Thomas , Vigano M. Alessandra , Ribeiro Carlos , Nussbaumer Ute , Grieder Nicole C. , Affolter Markus
Published: April 2021

A central theme during development and homeostasis is the generation of cell type-specific responses to the action of a limited number of extant signaling cascades triggered by extracellular ligands. The molecular mechanisms by which information from such signals are integrated in responding cells in a cell-type specific manner remain poorly understood. We have undertaken a detailed characterization of an enhancer that is regulated by DPP signaling and by the homeotic protein Labial and its partners, Extradenticle and Homothorax. The expression driven by this enhancer (lab550) and numerous deletions and point mutants thereof was studied in wild-type and mutant Drosophila embryos as well as in cultured cells. We find that the lab550 enhancer is composed of two elements, a Homeotic Response Element (HOMRE) and a DPP Response Element (DPPRE) that synergize. None of these two elements can reproduce the expression of lab550, either with regard to expression level or with regard to spatial restriction. The isolated DPPRE of lab550 responds extremely weakly to DPP. Interestingly, we found that the inducibility of this DPPRE is weak because it is tuned down by the action of a repressor element. This repressor element and an additional 50 bp element appear to be crucial for the cooperation of the HOMRE and the DPPRE, and might tightly link the DPP response to the homeotic input. The cooperation between the different elements of the enhancer leads to the segmentally restricted activity of lab550 in the endoderm and provides a mechanism to create specific responses to DPP signaling with the help of a HOX protein complex.