Skin cell nuclear transfers 99

Original explantremoved

Adult frog of \-nu strainas nuclear donor

Outgrowth ofepidermal cells

Parent of 1st transferrecipient eggs Enucleation of

recipient eggsFoot web outgrowthprove frog was 2-nu

Donor cells fornuclear transfer

1st nuclear transfer Cells trypsinizedand washed

Uncleaved Completely cleaved(70 V) Martially cleaved /c o/\

(25%)

Dissociated cells forserial transfer

I * ^ i / KJpZ*Parent of serial ti

1 recipient eggs

transferEnucleation ofrecipient eggs

Foot web outgrowthprove frog was 2-nu

Serial nuclear transfer

Uncleaved Completely cleaved(40/O Partially cleaved (30/0

(30%)

Nuclear transplant tadpole:l-nu diploid from nucleolus and chromosome counts

(present in 36% of serial clones)

Fig. 2. Plan of serial nuclear transfer experiments, using nuclei from adult skin celJs.Reasons for the various steps are explained in the text (p. 99). The percentages ofinjected eggs which cleave in different ways are those typically obtained with adultskin cell nuclei. The actual results of the experiments reported here are shown inTables 1 and 3.

The overall plan of our experiments is illustrated in Fig. 2. Serial transferswere carried out in all cases, and the following comments summarize thereasons for this. A serial nuclear transfer experiment is one in which a donornucleus is taken from an embryo which has itself resulted from a previous,first-transfer, experiment (Fig. 2). Most of the blastulae obtained from first

Nuclear transplantation, serial transplantation improves success, and enables the demonstration that cells do not lose information for differentiation (from the classic)

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http://youtu.be/3OuKuEmivug

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Somite differentiation productsPictures from Stockdale et al.

Pax3(dermomyotome,hypaxial muscle,dorsal neural tube)

Pax1(sclerotome)

Pax3

Pax1MyoD (muscle)

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Myogenin-lacZ transgene in E11.5 embryosKablar et al., 2003

Myf5−/−:MyoD−/−

Myf5−/−

MyoD−/−

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The activity of MyoD is coupled to cell-cycle control.

Growth factors induce proliferation, and activate cyclin dependent kinases. CDK inhibits MyoD activity by phosphorylation.

During differentiation, a positive feedback loop causes withdrawal from the cell cycle. High p21expression prevents re-entry into the cell cycle and activates the MyoD complex.

Proliferating Myoblast Differentiating myotube

Growth Factors

CDK/Cyclin MyoD

Proliferation

p21 (cyclin inhibitor

MyoD

CDK/Cyclin

co-activators

target (muscle)genes

Differentiation

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The belgian blue - a mutation in GDF8/myostatin

Myf5−/−:MyoD−/−

Myf5−/−

MyoD−/−

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http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=wing,bird&rid=dbio.figgrp.72Friday, April 20, 12

Brunet, L. J., McMahon, J. A., McMahon, A. P. & Harland, R. M. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 280, 1455-7 (1998).

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Patterning mechanisms controlling vertebrate limb development.Capdevila J, Izpisúa Belmonte JC.Annu Rev Cell Dev Biol. 2001;17:87-132. Review.

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http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=dbio.figgrp.3926

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K. Tosney Scanning EM of limb bud FGF8 expression

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A re-examination of proximodistal patterning during vertebrate limb development.Dudley AT, Ros MA, Tabin CJ.Nature. 2002 Aug 1;418(6897):539-44.

Spots of lineage trace in the early limb bud are restricted to subregions of the limb, illustrating some prepattern

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The AER is needed for outgrowth of the limb budFriday, April 20, 12

The ZPA defines the posterior to anterior axis of the limb

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The ZPA expresses Shh

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Functions of FGF signalling from the apical ectodermal ridge in limb developmentXin Sun, Francesca V. Mariani and Gail R. MartinNature 418, 501-508(1 August 2002)

Schematic of conditional FGF4 and FGF8 alleles, and the expression of these FGF genes in normal and mutant limb buds

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Functions of FGF signalling from the apical ectodermal ridge in limb developmentXin Sun, Francesca V. Mariani and Gail R. MartinNature 418, 501-508(1 August 2002)

mutant phenotype after removal of both FGF4 and FGF8

deletion of proximal limb elements after removal of FGF8 only

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http://www.ncbi.nlm.nih.gov/pubmed/9927590Friday, April 20, 12

5521E xpression and function of G remlin

dramatically enlarged skeletal elements following retrovirusinduced misexpression of bmp2 or bmp4 genes (Duprez et al.,1996a). Characteristically, in physiological conditions,prechondrogenic condensations exclude the superficialmesoderm subjacent to the ectodermal layer (Solursh, 1984).In this study, we have found that these non-chondrogenicregions correspond nicely to the zone of gremlin expression.Taking into account the high potential of exogenous Gremlinto block chondrogenesis in vivo, our results indicate thatGremlin has the function of confining chondrogenesis to thecentral core of the limb. In accordance with this possible roleof gremlin in the control of chondrogenesis, its expression inthe zone of implantation of the limb into the trunk may berelated to the formation of the shoulder girdle skeleton whichis also influenced by BMPs (Hofmann et al., 1998). It isinteresting to note that, unlike Gremlin, the BMP antagonistNoggin is expressed in the chondrogenic condensations andappears to control the growth and shape of the cartilages(Merino et al., 1998) rather than the establishment of theprechondrogenic condensations. Thus, while both Noggin andGremlin seem to be involved in the control of chondrogenesis,they may act in a complementary fashion rather than beingredundant signals.

The presence of gremlin in the dorsal and ventral mesodermmight be related to the establishment of the developing musclemasses in these regions. In vivo and in vitro studies indicatethat BMPs induce apoptosis of myogenic cells (Duprez et al.,1996b; Amthor et al., 1998) while at low concentration theymay regulate muscle differentiation (Amthor et al., 1998). Thedistribution of gremlin transcripts is compatible with a role of

this factor protecting the premyogenic cells from the apoptoticinfluence of BMPs. However, our findings indicate thatGremlin does not regulate early myogenic differentiation asdeduced from the lack of changes in the expression of MyoDfollowing Gremlin treatments.

The second period of gremlin expression in the limbcorresponds to the stages of formation of the digits (stages 27-32). During these stages, in addition to being expressed in thezones of feather formation and in the differentiating muscles,gremlin is observed in the interdigital mesenchyme. Majordifferences are appreciable in this period between the chick andduck leg. In the chick, only the most proximal region of theinterdigits exhibits a transitory expression of gremlin. Fromstage 30, coinciding with the onset of interdigital cell death,gremlin expression disappears from the chick interdigits.Unlike the chick, gremlin expression is maintained in theinterdigital mesoderm of the duck leg. The difference betweenthe webbed digits of the duck and the free digits of the chickis related to the extension of the areas of interdigital cell death(Saunders and Fallon, 1967). In addition, the role of BMPs incontrolling interdigital regression in the chick has beendemonstrated by a variety of experimental approaches (Zouand Niswander, 1996; Gañan et al., 1996; Yokouchi et al.,1996; Kawakami et al., 1996; Macias et al., 1997). However,surprisingly, the interdigital webs of the duck exhibit a patternof bmp gene expression virtually identical to that of the chick.(Laufer et al., 1997). Thus, the continued expression of gremlinin the duck interdigit observed here may serve to neutralizeinterdigital BMPs. In accordance with this interpretation, wehave observed that a duck-like syndactyly is induced in thechick by application of exogenous Gremlin in the interdigitalmesoderm. In addition, the presence of gremlin in the duckinterdigit may also explain the reduced expression of msxgenes in this species (Gañan et al., 1998). This is a significantfinding since msx-2 gene appear to be required in the apoptoticpathway mediated by BMPs (Graham et al., 1994; Gañan etal., 1998; Rodriguez-Leon et al., 1999).

The third period of gremlin expression in the limb covers thestages of maturation of the limb tissues once the anatomicalcomponents of the limb have been established. In this lateperiod of limb development, gremlin transcripts are found inthe differentiating perichondrium except in the zones of jointformation. This expression is coincident with that of bmp7(Macias et al., 1997) and may be related to the control ofcartilage growth and osteogenic differentiation by BMPs(Enomoto-Iwamoto et al., 1998). As mentioned above for theprevious period, gremlin is also expressed at these late stagesof development in the developing feathers, a process in whichBMPs play a central role (Jung et al., 1998).

In conclusion, the present study provides evidence for a keyrole of Gremlin as a mediator of the early signalling centersresponsible for limb outgrowth (AER and ZPA), whichmodulates the action of BMPs on growth, apoptosis and earlyskeletogenesis. In addition, Gremlin appears also involved inthe control of interdigital tissue regression and in later stagesin the regulation of the differentiation of the skeletal andmuscular limb tissues.

Sonia Pérez-Mantecón is acknowledged for technical assistance.This work was supported by grants from the DGICYT (PM95-0090;and PM96-0020). Finacial support form the Fundación Marqués de

Fig. 7. Expression of gremlin during the formation of the digits in theduck. (A-C) Interdigital expression of gremlin in the duck limb atdays 8.5 (A), 9 (B), and 10 (C) of incubation. (D) Duck leg bud atday 10 of incubation vital stained with neutral red showing the areasof interdigital cell death. Note that the distribution of cell deathcorresponds with the most distal mesenchyme of the interdigitlacking gremlin expression (compare C and D).

Development. 1999 Dec;126(23):5515-22.

The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb.Merino R, Rodriguez-Leon J, Macias D, Gañan Y, Economides AN, Hurle JM.

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