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Biomedical subjects

L Niswander

Publications and source records attributed to L Niswander.

At least 19 recordsLinked to original sources
PubMed2001

Early steps in limb patterning and chondrogenesis.

The interplay of a number of signalling molecules coordinates growth and patterning of the early embryonic vertebrate limb along the three axes. An unresolved question is how this information translates into proper positioning and patterning of the skeletal elements. More is known about how these elements develop. Cells first form precartilaginous condensations and then subsequently differentiate into chondrocytes. This provides a cartilage template that will ultimately be ossified to give rise to the bony skeleton. Several studies support a role for the bone morphogenetic proteins (BMPs) as extracellular signals that regulate early steps of limb chondrogenesis (cartilage formation). However, they have not clarified the step(s) at which BMPs act, and no genetic evidence is available to date. Here, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. We find that BMP signalling is necessary for the formation of precartilaginous condensations, their differentiation into chondrocytes, and for maintenance of chondrogenesis. These results also indicate that Noggin could be clinically useful to treat diseases involving ectopic cartilage formation.

Animals↗
PubMed2001

Expression of slit-2 and slit-3 during chick development.

The Drosophila and vertebrate slit proteins have been characterized as secreted chemorepellents recognized by the robo receptor proteins that function principally for the guidance of neuronal axons and neurons. slit genes are also expressed in the limb. To provide a basis for the determination of slit functions in the limb we have isolated and characterized the expression of chick slit-2 and slit-3 in the developing limb and other tissues of the chick embryo. Both genes share similar expression profiles in the chick embryo when compared to that of their mammalian homologues, particularly in the neural tube. In the limb, their expression patterns suggest their involvement in many aspects of limb development. In the early limb bud, slit-2 is expressed in the peripheral mesenchyme and invading muscle precursors, while slit-3 expression is restricted to the future chondrogenic core of the limb bud. At later stages, both slit genes are expressed in interdigital mesenchyme, in inner periosteal cells, and in mesenchyme immediately radial to the periosteum and under the epidermis. slit-3 is also expressed in proliferating chondrocytes during cartilage development, while slit-2 is expressed in later muscle masses and peripherally to joints in the autopod.

Animals↗
PubMed2001

The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.

The identification and characterization of DNA sequences necessary for proper gene expression have provided insights into gene regulation and generated tools useful for further experimentation. Studies of developmentally regulated genes have demonstrated how transcription factors interact at enhancers to generate restricted patterns of expression during embryogenesis. In vertebrates, the pursuit of such studies has relied on the generation of transgenic mice and thus has been limited by the time and expense required generating and characterizing these mice. The recently developed technique of in ovo electroporation allows the rapid introduction of exogenous DNA into developing chicken embryos. Here we have used this technique to introduce DNA containing murine enhancer/reporter constructs into cells of the chicken neural tube, resulting in appropriate expression of the reporter. This technique has the potential to greatly reduce the effort involved in the study of vertebrate enhancers. Furthermore, we have characterized factors such as timing of electroporation, concentration of DNA, and choice of basal promoters and found that they can influence the degree to which expression of enhancer constructs reflects endogenous gene expression.

Animals↗
PubMed2001

BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb.

Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the apical ectodermal ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, we demonstrate that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. Our results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and our results suggest that MSX acts downstream of BMP in AER induction. We propose that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively.

Animals↗
PubMed2000

BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes.

Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205-217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes.

Animals↗
PubMed2000

Plzf regulates limb and axial skeletal patterning.

The promyelocytic leukaemia zinc finger (Plzf) protein (encoded by the gene Zfp145) belongs to the POZ/zinc-finger family of transcription factors. Here we generate Zfp145-/- mice and show that Plzf is essential for patterning of the limb and axial skeleton. Plzf inactivation results in patterning defects affecting all skeletal structures of the limb, including homeotic transformations of anterior skeletal elements into posterior structures. We demonstrate that Plzf acts as a growth-inhibitory and pro-apoptotic factor in the limb bud. The expression of members of the abdominal b (Abdb) Hox gene complex, as well as genes encoding bone morphogenetic proteins (Bmps), is altered in the developing limb of Zfp145-/- mice. Plzf regulates the expression of these genes in the absence of aberrant polarizing activity and independently of known patterning genes. Zfp145-/- mice also exhibit anterior-directed homeotic transformation throughout the axial skeleton with associated alterations in Hox gene expression. Plzf is therefore a mediator of anterior-to-posterior (AP) patterning in both the axial and appendicular skeleton and acts as a regulator of Hox gene expression.

Animals↗
PubMed1999

BMPs negatively regulate structure and function of the limb apical ectodermal ridge.

The apical ectodermal ridge (AER), a transient specialized epithelium at the distal limb tip, is essential for vertebrate embryonic limb outgrowth along the proximodistal axis. Among all the molecules expressed in the AER, only the Fibroblast Growth Factors (FGFs) have been shown to substitute for its function in limb outgrowth. After specification of the skeletal progenitors is complete, the AER regresses, having fulfilled its function. However, the cellular processes underlying AER regression remain largely unclear, and the molecular ones, totally unknown. Members of the Bone Morphogenetic Protein (BMP) family are expressed in the AER throughout its life and in the mesenchyme. Our studies using misexpression of Noggin, a BMP inhibitor, reveal an unsuspected role for BMPs in the negative regulation of Fgf expression and AER function. We find that BMPs limit limb outgrowth by promoting AER regression, as BMP inhibition results in persistence of the AER, prolonged Fgf expression and excess soft-tissue growth. In addition, the Noggin misexpression studies uncover an earlier role for BMPs in repression of AER function. Noggin overexpression results in extension of the AER anteriorly and loss of AER asymmetry. We show that overall the AER becomes taller, and its anterior half becomes more similar to a normal posterior AER. In addition, Fgf4 transcripts, which are usually restricted to the posterior half of the AER, are now also expressed anteriorly. Moreover, ectopicFgf4 expression is induced independently of Sonic Hedgehog, contrary to current models of Fgf4 regulation in the limb. Our studies also provide insight into the activity of the hypothesized apical ectodermal maintenance factor (AEMF), which is thought to maintain the tall shape of the posterior part of the AER. Our work shows that the AER is negatively regulated by BMP.

Animals↗
PubMed1999

Delta-1 negatively regulates the transition from prehypertrophic to hypertrophic chondrocytes during cartilage formation.

Endochondral bone development begins with the formation of a cartilage template. Chondrocytes within this template undergo a progressive program of maturation from proliferative to prehypertrophic chondrocytes to hypertrophic chondrocytes. The progression of cells through these steps of differentiation must be carefully controlled to ensure coordinated growth. Because the Delta/Notch signaling system is known to regulate cell fate choices, we sought to determine if these molecules might be involved in the progressive cell fate decisions that chondocytes undergo. Here we demonstrate in the chick that Delta/Notch signaling negatively regulates progression from the prehypertrophic to hypertrophic state of differentiation. Delta-1 is expressed specifically in the hypertrophic chondrocytes while Notch-2 is expressed in chondrocytes at all stages. Misexpression of Delta-1 using a replication-competent retrovirus blocks chondrocyte maturation. Prehypertrophic cells form normally but do not undergo differentiation to hypertrophic cells, resulting in shortened skeletal elements that lack ossification. We conclude that Delta-1 acts during chondrogenesis to inhibit the transition from prehypertrophic chondrocytes to hypertrophic chondrocytes, thus defining a novel mechanism for the regulation of the chondrocyte maturation program. In addition, these results reveal a new role for Delta/Notch signaling in regulating the progression to a terminally differentiated state.

Animals↗
PubMed1999

The surface ectoderm is essential for nephric duct formation in intermediate mesoderm.

The nephric duct is the first epithelial tubule to differentiate from intermediate mesoderm that is essential for all further urogenital development. In this study we identify the domain of intermediate mesoderm that gives rise to the nephric duct and demonstrate that the surface ectoderm is required for its differentiation. Removal of the surface ectoderm resulted in decreased levels of Sim-1 and Pax-2 mRNA expression in mesenchymal nephric duct progenitors, and caused inhibition of nephric duct formation and subsequent kidney development. The surface ectoderm expresses BMP-4 and we show that it is required for the maintenance of high-level BMP-4 expression in lateral plate mesoderm. Addition of a BMP-4-coated bead to embryos lacking the surface ectoderm restored normal levels of Sim-1 and Pax-2 mRNA expression in nephric duct progenitors, nephric duct formation and the initiation of nephrogenesis. Thus, BMP-4 signaling can substitute for the surface ectoderm in supporting nephric duct morphogenesis. Collectively, these data suggest that inductive interactions between the surface ectoderm, lateral mesoderm and intermediate mesoderm are essential for nephric duct formation and the initiation of urogenital development.

Animals↗
PubMed1998

Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis.

In Drosophila, the Dorsal protein establishes the embryonic dorso-ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-kappaB), is vital for the formation of the proximo-distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-kappaB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-kappaB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-kappaB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-kappaB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

Adenoviridae↗
PubMed1998

Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures.

Previous work has demonstrated that the bone morphogenetic proteins (BMP)-2, BMP-4, and BMP-7 can promote the development of tyrosine hydroxylase (TH)-positive and catecholamine-positive cells in quail trunk neural crest cultures. In the present work, we showed that mRNA for the type I bone morphogenetic protein receptor IA (BMPR-IA) was present in neural crest cells grown in the absence or presence of BMP-4. We have used a replication-competent avian retrovirus to express a constitutively active form of BMPR-IA in neural crest cells in culture. Cultures grown in the absence of BMP-4 and infected with retrovirus containing a construct encoding this activated BMPR-IA developed five times more TH-immunoreactive and catecholamine-positive cells than uninfected control cultures or cultures infected with virus bearing the wild-type BMPR-IA cDNA. The number of TH-positive cells which developed was dependent on the concentration of virus bearing the activated receptor cDNA used in the experiments. Most TH-positive cells which developed also contained viral p19 protein. Total cell number was not affected by infection with the virus containing the activated receptor construct. The effect of the activated receptor was phenotype-specific since infection with the virus bearing the activated receptor cDNA did not alter the number or morphology of microtubule-associated protein (MAP)2-immunoreactive cells, which are distinct from the TH-positive cell population. These findings are consistent with the observation that MAP2-positive cells are not affected by the presence of BMP-4. Taken together, these results suggest that activity of BMPR-IA is an important element in promoting the development of the adrenergic phenotype in neural crest cultures.

Adrenergic Fibers↗
PubMed1998

Disruption of scale development by Delta-1 misexpression.

Notch and its ligands are molecules known to be involved in many cell fate decisions. Due to the proposed role of this signaling system in the choice between feather and smooth skin fate in the embryonic chick, we sought to test whether Notch signaling may also play a role in determining scale fate. Notch-1 and -2, Serrate-1 and -2, and Delta-1 are expressed in the scales throughout their development. Misexpression of chick Delta-1 using a replication-competent retrovirus results in regions of scale loss. In addition, feather buds are often observed on some of the scuta scales. These results suggest a model in which Delta can act as an inhibitor of skin appendage fate as well as a promoter of feather fate.

Animals↗
PubMed1998

Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development.

We have recently shown in mice that four members of the T-box family of transcription factors (Tbx2-Tbx5) are expressed in developing limb buds, and that expression of two of these genes, Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. In this report, we investigate the role of these genes in limb specification and development, using the chick as a model system. We induced the formation of ectopic limbs in the flank of chick embryos to examine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subsequently develop. We found that, whereas bud regions expressing Tbx4 developed characteristic leg structures, regions expressing Tbx5 developed characteristic wing features. In addition, heterotopic grafts of limb mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, retained autonomous expression of the appropriate, limb-specific T-box gene, with no evidence of regulation by the host bud. Thus there is a direct relationship between the identity of the structures that develop in normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, we employed several other embryological manipulations. By surgically removing the apical ectodermal ridge (AER) from either wing or leg buds, we found that, in contrast to all other genes implicated in the patterning of developing appendages, maintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity (ZPA). By generating an ectopic ZPA, by grafting a sonic hedgehog (SHH)-expressing cell pellet under the anterior AER, we found that Tbx2 expression can lie downstream of SHH. Finally, by grafting a SHH-expressing cell pellet to the anterior margin of a bud from which the AER had been removed, we found that Tbx2 may be a direct, short-range target of SHH. Our findings suggest that these genes are intimately involved in limb development and the specification of limb identity, and a new model for the evolution of vertebrate appendages is proposed.

Animals↗
PubMed1998

A new role for Notch and Delta in cell fate decisions: patterning the feather array.

Chick embryonic feather buds arise in a distinct spatial and temporal pattern. Although many genes are implicated in the growth and differentiation of the feather buds, little is known about how the discrete pattern of the feather array is formed and which gene products may be involved. Possible candidates include Notch and its ligands, Delta and Serrate, as they play a role in numerous cell fate decisions in many organisms. Here we show that Notch-1 and Notch-2 mRNAs are expressed in the skin in a localized pattern prior to feather bud initiation. In the early stages of feather bud development, Delta-1 and Notch-1 are localized to the forming buds while Notch-2 expression is excluded from the bud. Thus, Notch and Delta-1 are expressed at the correct time and place to be players in the formation of the feather pattern. Once the initial buds form, expression of Notch and its ligands is observed within each bud. Notch-1 and -2 and Serrate-1 and -2 are expressed throughout the growth and differentiation of the feathers whereas Delta-1 transcripts are downregulated. We have also misexpressed chick Delta-1 using a replication competent retrovirus. This results in induction of Notch-1 and-2 and a loss of feather buds from the embryo in either large or small patches. In large regions of Delta-1 misexpression, feathers are lost throughout the infected area. In contrast, in small regions of misexpression, Delta-1 expressing cells differentiate into feather buds more quickly than normal and inhibit their neighbors from accepting a feather fate. We propose a dual role for Delta-1 in promoting feather bud development and in lateral inhibition. These results implicate the Notch/Delta receptor ligand pair in the formation of the feather array.

Animals↗
PubMed1997

Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage.

The bone morphogenetic proteins (BMPs), TGF beta superfamily members, play diverse roles in embryogenesis, but how the BMPs exert their action is unclear and how different BMP receptors (BMPRs) contribute to this process is not known. Here we demonstrate that the two type I BMPRs, BMPR-IA and BMPR-IB, regulate distinct processes during chick limb development. BmpR-IB expression in the embryonic limb prefigures the future cartilage primordium, and its activity is necessary for the initial steps of chondrogenesis. During later chondrogenesis, BmpR-IA is specifically expressed in prehypertrophic chondrocytes. BMPR-IA regulates chondrocyte differentiation, serving as a downstream mediator of Indian Hedgehog (IHH) function in both a local signaling loop and a longer-range relay system to PTHrP. BMPR-IB also regulates apoptosis: Expression of activated BMPR-IB results in increased cell death, and we showed previously that dominant-negative BMPR-IB inhibits apoptosis. Our studies indicate that in TGF beta signaling systems, different type I receptor isoforms are dedicated to specific functions during embryogenesis.

Animals↗
PubMed1997

Limb mutants: what can they tell us about normal limb development?

Classical mutations and those that derive from gene targeting have provided an important resource to explore the molecular control of vertebrate limb development. Recent studies have combined molecular analysis of limb mutants with embryological approaches to understand the regulation of limb patterning and growth.

Animals↗