human molecular embryogenesis: an overview

PERSPECTIVES IN PEDIATRIC PATHOLOGY

Human Molecular Embryogenesis:An Overview

LUC LAURIER OLIGNY

Department of Pathology and Cellular Biology, Faculty of Medicine, University of Montreal, and Hopital Sainte-Justine, 3175, Chemin de la Cote Ste-Catherine, Montreal, Quebec, Canada H3T 1C5

Received December 15, 2000; accepted February 15, 2001.

ABSTRACTMolecular embryology is a rapidly evolving field of greatcomplexity. This overview is primarily for the newcomerto this field, in an attempt to demystify the processes bywhich a human single-celled zygote eventually forms anembryo. Although all embryonic cells share the samegenetic information, they differentiate according to thebasic plan dictated not only by multiple families of tran-scription factors to silence some genes and activate oth-ers but also through DNA methylation, histone acetyla-tion, and heterochromatinization. Regional expressionof various transcription factors causes embryos to estab-lish primary embryonic axes. Once the basic body plan isestablished, the region-specific diversity becomes pro-gressively finer, and each cell eventually develops a “mo-lecular address” characterized by the expression of spe-cific genes. The overview is divided into two main parts:embryonic cell growth and morphogenesis. At thepresent time, more is known about the details of molec-ular regulation of the embryonic growth than aboutmorphogenesis.

Key words: molecular embryogenesis, morphogenesis,embryonic cell growth

INTRODUCTIONThis article is intended to be a clinically orientedbasic overview of molecular embryology. It as-sumes little prior molecular genetics background,as the concepts are explained throughout. Themain goal of the article is to provide a molecular

framework for an understanding of the basicmechanisms used by a unicellular zygote to forman embryo. To achieve this goal, the complexity ofthese mechanisms is illustrated through severalknown dynamic cascades of events regulated by alarge number of factors that determine cell prolif-eration, cell differentiation, programmed celldeath, and morphogenesis of the embryo.

Traditional embryology, both descriptive andexperimental, is characteristically concerned witheither the whole developing embryo or its specificparts in relation to the development of the wholeembryo. Molecular embryology is less inclusive, asit usually focuses on differential expression ofgenes and the effects of their transcripts in partic-ular cells or tissues. Transcription of most genesproduces a messenger RNA (mRNA) that is trans-lated from the four-letter alphabet of the nucleicacids (nucleotides) to the 20-letter alphabet of theprotein (amino acids), using the genetic code (Fig.1). Proteins produced within the cells determinecellular metabolism, behavior, polarity, shape, ad-hesiveness, and receptivity to extracellular signals.In addition, some proteins have a role in binding toDNA. Genes that encode such proteins are referredto as regulatory genes and the proteins themselvesare known as transcription factors, because they

Pediatric and Developmental Pathology 4, 324–343, 2001

DOI: 10.1007/s10024-001-0033-2

Pediatric and Developmental Pathology

© 2001 Society for Pediatric Pathology

control (up-regulate and down-regulate) transcrip-tional activity of the gene to which they havebound. In essence, genes work in hierarchies, withregulatory genes controlling the expression ofdownstream genes and communicating amongthemselves. Such genetic cascades and signalingpathways are pivotal, not only in maintaining thenormal life of the cell but also during embryonicdevelopment in determining cell fate [1].

Within the increasingly multicellular embryo,cell interactions and inductions are initiated. Theseare based on the interactions of multiple proteinswith groups of cells to generate a progressivelyhigher order of complexity. During embryonic devel-opment, many of these higher-order events takeplace in the absence of direct genetic control, al-though they are the inevitable consequences of ge-netic specification. This type of regulation is knownas epigenetic.

Descriptive human embryology has providedthe names for most processes happening from fer-tilization until birth (cleavage, gastrulation, germlayer formation, organogenesis), but the complete

molecular mechanisms even for relatively simple

events, such as sperm–egg fusion during fertiliza-

tion, the mechanics of pronuclear migration, and

the timing mechanism of the first zygotic division

after fertilization, are not fully understood. As the

complexity of embryonic development increases,

cell differentiation, the coordination of gastrula-

tion movements, and mechanisms of directed cell

locomotion are considered during morphogenesis,

the molecular cascades are even more nebulous

[2,3].

Embryonic development starting from a single

cell is characterized by extensive cell growth, which

involves proliferation, cell death, and generation of

cellular diversity (differentiation). This growth is ac-

companied by the creation of a specific form and

order by organizing the differentiated cells into tis-

sues and organs (morphogenesis). In humans, the

complete embryo (3 cm in length) is formed within

63 days after fertilization. The remaining time of the

human pregnancy (244 days) is dedicated to fetal

growth, which means a further increase in size of all

Figure 1. Gene expression is regulated by the coordi-nated action of transcription factors (TF) which bind con-trol regions in DNA. First, TFs bind the promoter, stimu-lating the TATA box binding protein (TBP) to bind theTATA box, a region rich in thymidine and adenine. Thiscauses multiple proteins to coalesce with the TBP to forma co-activator complex. The formation of the co-activatorcomplex is modulated by the binding of multiple tran-

scription factors onto enhancers and silencers, to facili-tate or inhibit its assembly. Once this machinery is inplace, RNA polymerase starts transcription of RNA; notethat binding of silencers by TFs inhibits the formation ofthe co-activator complex, and it is the cumulative effectof all the TFs on silencers and enhancers that will deter-mine the extent of a gene’s transcription, from negligi-ble to abundant.

HUMAN MOLECULAR EMBRYOLOGY 325

organs and tissues already present in the embryo,and their maturation.

GLOSSARYApoptosis: Genetically programmed death of acell. It is characterized by the intracytoplasmic andintranuclear activation of an enzymatic cascadethat digests the cell from inside-out. It results incellular “dropout” without an associated inflam-matory reaction.Determination: Commitment of the cell to differ-entiation. It can be achieved either by cytoplasmicsegregation of determinative molecules during em-bryonic cleavage or by embryonic induction.Differentiation: Generation of cellular diversitysuch as the development of specialized cell typesfrom a single fertilized egg.Embryonic induction: Interaction of cells or tis-sues that determines the fates of one or both par-ticipants.Enhancers/silencers: DNA sequence elementsthat increase or decrease the level of transcriptionof a gene are called enhancers or silencers, respec-tively.Epigenetic: All processes relating to the expres-sion of genes that are determined by events beyondthe level of genetic information.Exons: The regions of the gene that are tran-scribed into messenger RNA, and subsequentlyusually translated into a functional protein prod-uct.Gastrulation: Process by which the embryo ac-quires two or more germ layers. In human em-bryos, ectoderm, endoderm, and mesoderm areformed.Heterochromatin: A condensed chromatin withlate replication and transcriptional inactivity in thecompacted state.Intron: The DNA sequence intervening betweentwo exons. Introns are spliced out during the pro-cessing of the messenger RNA into a mature mes-sage.Morphogen: A substance that, in a concentration-dependent manner, determines the future identityof a cell. Morphogens can act within a cell (e.g.,transcription factors) or between cells (e.g., cellsignaling molecules).Morphogenesis: Process of bringing cell popula-tions together for new inductive interactions and

for building complex three-dimensional structuresout of simple epithelial sheets and mesenchymalcell masses.Morphogenetic field: An embryonic area whosecells can co-operatively bring about a distinctstructure or set of structures with the help of signalsubstances such as an inducer or morphogen. Mor-phogenetic fields define developmental potencies.Nucleosome: The human genome of 3 3 109 basepairs (bp) is longer than one meter when unrav-eled; however, it is compacted in a nucleus, whichis only 10 mm in diameter on average. This feat isachieved partly through wrapping DNA aroundspecial proteins called histones. A nucleosome is aunit that comprises this protein core and the seg-ment of DNA between two histone cores. The DNAsegment contained in a nucleosome is wrappedtwice around each histone core and there are gen-erally between 180 and 200 nucleotides betweeneach histone core (i.e., within each nucleosome).Promoter: The promoter region is defined as thesequences that are involved in the initiation oftranscription. This region can generally be foundwithin 300 bp of the transcription start site. Thisregion may contain several DNA elements that in-clude the TATA box, the CCAAT box, and otherrelatively generic sequences that determine wherethe RNA polymerase binds and transcriptionstarts.Signaling molecules: Effective in extremelyminute amounts these molecules are involved inembryogenesis as determination factors, morpho-gens, inducers, growth factors, and differentiationfactors.Splicing: Process involving the molecule of mes-senger RNA through which introns are removedand exons are joined to produce a mature message.Transcription factors: Molecules (proteins andother molecules such as retinoic acid or steroidhormone receptors) that bind DNA directly in theregions of promoter, enhancer, and/or silencer toactivate or inhibit the expression of gene(s) undertheir control.

EMBRYONIC CELL GROWTHProcesses of embryonic cell growth, starting froma single fertilized cell known as a zygote, consist ofextensive and accurately orchestrated cell prolifer-ation, differentiation, and apoptosis.

326 L.L. OLIGNY

Cell proliferationEmbryonic cell proliferation (recurring cell divi-sions) requires interactions of many cyclin-depen-dent kinases regulating various parts of the cellcycle as well as interactions with signaling mole-cules, inhibitors, and receptors. Proliferation mustbe precisely regulated and closely connected with acontrol of differentiation. The activation and inac-tivation of proliferation genes is synchronized withboth cell differentiation and programmed celldeath [4]. During the 4 weeks following fertiliza-tion, the number of cells doubles every 2 to 4 days,under the control of these genes. Regional differ-ences in growth are dictated by the expression ofthese genes and reflect the cumulative effect ofproliferation and the ongoing loss of cells throughprogrammed cell death.

Cell differentiationAll cells of an embryo, despite their various shapes,sizes, and function, have the same genetic infor-mation stored in their nuclei. Cellular differentia-tion is characterized by the permanent activationof certain genes and the inactivation of other genesto allow a more undifferentiated cell to specialize,(e.g., the evolution of a pluripotent mesoblastic cellinto a muscle cell). Undifferentiated embryonicstem cells often give rise to one cell as undifferen-tiated as itself and to one more differentiateddaughter cell. Daughter cells become progressivelyfully differentiated as they continue to divide. Thusdifferentiated somatic cells have permanently in-activated a large subset of genes [5].

During the formation and normal earlygrowth of an embryo, all genes are potentially ac-tive and available for transcription. Even in theblastocyst, the inner cell mass cells are still omnip-otent, as cellular separation at this stage results inthe formation of monozygotic twins. The molecu-lar cascades that control cellular differentiationare not fully understood, but some basic principleshave emerged. (1) Genes are not expressed untiltheir promoters are activated by transcription fac-tors. (2) Repression of gene transcription is ini-tially achieved through the inhibition of transcrip-tion factors on genes’ promoters which thenbecome methylated and permanently inactivated.(3) As methylation is passed from one cell to all itsdaughter cells, gene inactivation is generally irre-

versible. (4) Other means of stable gene regulation,histone acetylation, and heterochromatinizationare also passed on to daughter cells. (5) Develop-mentally important genes can often activate theirown promoter (positive feedback). Thus a gene isperpetually activated by all of the descendants ofthe cell that initially expressed this gene (whichagain leads to differentiation being generally irre-versible). (6) Differentiation and mitotic activityare inversely proportional (the greater the differ-entiation of a cell population, the lower its prolif-eration rate) [6].

DNA methylation

DNA methylation inhibits the expression of genesthat are not needed by a given cell. This mecha-nism allows differentiation—i.e., a cell that willdifferentiate into a hepatocyte must inhibit all thegenes responsible for a mesenchymal differentia-tion, and this inhibition must be permanent—thatis, transmitted to all the daughter cells of thatprogenitor cell. DNA methylation, which is accom-plished by DNA-methyltransferases, occurs on thecytosine residues of CG (also called CpG) dinucle-otides. During the process of methylation, cytosineis methylated into 5-methyl-cytosine (m5C). In hu-mans, approximately 80% of CpG are methylatedwhile only 3–8% of all cytosines are methylated.Once a CpG becomes methylated in a cell, it willremain methylated in all its descendants [7].

Methylation of promoters inhibits their rec-ognition by transcription factors and RNA poly-merase, as methylated cytosines preferentiallybind to a protein called methyl cytosine bindingprotein, or MeCP (Fig. 2). When a promoter regionnormally recognized by an activating transcriptionfactor is methylated, its transcription will be inhib-ited. Some transcription factors are exquisitelysensitive to CpG methylation: the methylation of asingle CpG can activate or repress (in an on–offswitch-like fashion) the transcription of their sub-ordinate gene. In other promoters, the effect ofCpG methylation is gradual—the larger the num-ber of methylated CpGs, the greater the inhibitionof transcription. In some instances, when an inac-tive gene must be reactivated, nuclear demethy-lases will remove the methyl groups from its pro-moter.


Methylation of promoters is probably one ofthe foremost mechanisms responsible for cellu-lar differentiation during embryogenesis: thetranscription of unwanted genes is eliminated bymethylation of their promoters. As oocytes andspermatozoa are more differentiated than thetotipotent cells of the early embryo, the DNA ofmorula (16-cell embryo, third day postconcep-tion) undergoes global demethylation. CpGs aredemethylated on a large scale, thus reactivatingthe near-totality of the genome (a few genes es-cape this demethylation, e.g., the genes subjectto genomic imprinting). Subsequently, as cellsstart differentiating, the promoters of genes in-volved in this differentiation will become meth-ylated according to a strict sequence dependingon each cell type.

Histones and acetylation

Until recently, histones were thought to have anexclusively mechanical and structural role, to pro-tect DNA from injurious agents and to allow DNAcompaction. We now know that histones also playa major role in the control of transcription, expos-ing or hiding DNA sequences specifically recog-nized by RNA-polymerases and transcription fac-tors.

All four different histone proteins are subjectto acetylation of their positively charged lysines;

acetylation of each lysine decreases the electricalcharge of a histone by one electron. When histoneshave $3 acetylated lysines, they are referred to asbeing hyperacetylated, whereas they are said to behypoacetylated when containing #1. Histone acet-ylation modulates nucleosome formation and DNAcompaction into chromatin [8].

A gene’s rate of transcription is proportionalto the acetylation of its histones. Acetylation playsan important role in the interaction of nucleo-somes with DNA, influencing the secondary andtertiary structure of DNA, thus modulating the ef-ficiency with which RNA-polymerase can interactwith promoters to initiate transcription (Fig. 3).Transcriptionally active genes are often hyper-acetylated, and certain transcription factors con-tain acetylases to acetylate the histones of the pro-moters that they control. Histone acetylation andDNA methylation seem to be closely interrelated ata molecular level. The inactive methylated X chro-mosome in female embryos also shows hypoacety-lation of its histones.

Heterochromatinization through polycomb proteins

Polycomb proteins (Pcs) belong to a large family ofgenes called the Polycomb group. Pcs interact withchromatin through their binding of transcriptionfactors, in regions distant from the genes that theycontrol.

Figure 2. Effect of CG methylation on DNA recognitionby transcription factors. Many TFs are methyl-sensitive:they recognize and bind only unmethylated cytosines.Methyl-cytosine binding proteins (MeCP, such as MeCP-1and MeCP-2) specifically bind methyl-cytosines, prevent-ing TFs from binding to these sites. In addition, MeCPsbind MeCP-binding proteins (MeCP-BP, such as the tran-scriptional co-repressor SIN3A and the histone deacety-lases HDAC-1 and HDAC-2) to inhibit transcription.

Figure 3. Role of histones in gene transcription. A:Nucleosomes are DNA–histone complexes. Regions withactive transcription, such as promoters and enhancers ofnonsilenced genes, are usually free of histones, allowingoptimal recognition of these sequences by transcriptionfactors. Transcriptionally silenced regions with tightlypacked nucleosomes are known as histone-rich hetero-chromatin. B: Acetylation of histones changes the chro-matin configuration. This regulates binding of transcrip-tion factors, and thus gene expression.

328 L.L. OLIGNY

Once bound to DNA, Pcs attract and bindother Pcs, thus forming a Pc polymer (Fig. 4). Oncea Pc binds a gene promoter, the descendants ofthat cell will all have a Pc bound to the sameregion, resulting in the permanent silencing of thatregion. If many adjacent sites are recognized byPcs, the different Pcs will interact with each other,to form a large polymer linking the whole region.This polymerization folds and compresses DNA,altering the secondary and tertiary structure ofchromatin, causing it to become transcriptionallyinactive heterochromatin. Gene silencing by het-erochromatinization through Pcs contributes sig-nificantly to persistent and heritable embryonicdetermination and differentiation in Drosophila[9,10]. Their role in vertebrates embryogenesis hasnot yet been established.

Transcription factors

Transcription factors are molecules that bind tothe gene regulatory region to activate or inhibitthe expression of gene(s) under their control.This action is generally mediated through a di-rect interaction with the transcriptional controlmachinery (promoters, enhancers, silencers, and

protein–protein interactions, including RNApolymerase) of those genes. A large number oftranscription factors can interact simultaneouslywith this machinery, and it is the cumulativeeffect of all the activating and inhibiting forcesof these transcription factors that determines towhich extent a gene will be transcribed intomRNA [11].TRANSCRIPTION FACTOR FAMILIES. Many different fam-ilies of transcription factors are known (Table 1). Itis increasingly realized that mutations of thesetranscription factors are responsible for a numberof diseases; furthermore, a variety of different mu-tations within any one of the genes that they reg-ulate can result in a specific embryonic phenotypeeffect.

Transcription factors are activated at differ-ent times during embryogenesis. They interactwith one another, and it is the cumulative effect oftranscription factors at the level of gene promoters,enhancers, and silencers that determines the levelof expression of their subordinate genes. Some ofthe major transcription factor families are listedbelow.

Helix-loop-helix (HLH). This family of tran-scription factors owes its name to these factors’tertiary structure (Fig. 5). Some members of thisfamily control the expression of cell adhesion mol-ecules (CAMs; to be discussed later), thus modu-lating intercellular interactions, as well as the in-teraction between cells and the extracellularmatrix. The cell–mesenchyme interaction plays amajor role in the induction of morphogenesis.

Basic helix-loop-helix (bHLH). These pro-teins’ structure resembles that of other HLH. How-ever, to be active, they must form dimers at thelevel of genes’ promoters. “Basic” refers to pH asthe basic portion of these proteins binds acidicDNA. MYF-3 and MYF-5 belong to this family oftranscription factors.

PAX (Paired box). These transcription fac-tors are characterized by the presence of a re-peated (paired) DNA domain and homeodomain.Nine PAX genes are known (Fig. 6). The differentPAX genes contain various combinations of paireddomains, with homeodomains (or their parts) anda sequence called octapeptide (OP). Most PAXgenes appear to be involved in morphogenesis. Themolecular factors controlling the expression of in-

Figure 4. Polycomb and heterochromatinization. Pro-teins of the polycomb protein (Pc) family can recognizeand bind specific DNA-associated proteins. It is throughthis interaction, rather than through a specific Pc chro-modomain–DNA interaction, that Pcs mediate their ef-fect. Pcs bound to chromatin have a tendency to poly-merize and bind adjacent Pcs, so as to compact the DNAunder their control. This compacted DNA is thus perma-nently inactivated. It has been shown that Pcs serve toperpetuate transcription inhibition on previously silencedDNA rather than to be the direct cause of this inhibition.


dividual PAX genes are poorly understood, andonly rare target genes controlled by these tran-scription factors are known.

For example, PAX-3 is initially expressed byall cells of embryonic somites. Then, only the myo-blasts of somitic origin express PAX-3 and the ex-pression of PAX-3 by other somitic cells is re-

pressed. Finally, PAX-3 is expressed only bymyoblasts destined to populate the limbs. PAX-6 isanother example of the morphogenetic action ofPAX genes: PAX-6 initiates the development of theeye. Without PAX-6 expression, there is no attemptto form an eye. PAX-6 has been extremely wellconserved through evolution, both at the aminoacid sequence and at the functional level. In Dro-sophila, the ectopic expression of PAX-6 is suffi-cient to stimulate the perfect development of an

Table 1. Inherited human disorders due to mutations in genes encoding transcription factors

Nuclear receptors (family of zinc fingers)

Androgen receptor (AR): androgen insensitivity syndromes, and spinal/bulbar muscular atrophy

Estrogen receptor (ER): estrogen resistance

Glucocorticoid receptor (GR): glucocorticoid resistance

Thyroid hormone receptor b (TRb): thyroid hormone resistance

Vitamin D receptor (VDR): hereditary vitamin D resistant rickets type II

DAX1: X-linked adrenal hypoplasia congenita, dosage-sensitive sex reversal

Other zinc fingers

Wilms tumor 1 (WT1): WAGR syndrome, Denys-Drash syndrome

GLI-3: Grieg cephalopolysyndactyly syndrome, Pallister-Hall syndrome

PAX

PAX2: optic nerve coloboma and renal hypoplasia

PAX3: Waardenburg syndrome types I and III, craniofacial-deafness-hand syndrome

PAX6: aniridia, Peter’s anomaly, isolated foveal hypoplasia, autosomal dominant keratitis

hHLH

MITF: Waardenburg syndrome type II

TWIST: Saethre-Chotzen syndrome

Modified from Semenza [11].

Figure 5. Helix-loop-helix (HLH) type transcription fac-tors. The diagram to the left shows the structure of theHLH protein. The helical portions are tightly coiled spiralsof amino acids, whereas the loop joins the two helices toprovide optimal DNA interaction. The diagram to theright shows how the HLH protein binds a major groove ofthe DNA molecule. Subtle changes in the amino acid se-quence of the recognition helix causes HLH to recognizedifferent DNA sequences, and to thus bind and activateor repress the control sequences of a whole battery ofdifferent genes.

Figure 6. Structure of human PAX proteins. These pro-teins can contain a DNA-binding octapeptide (OP), a full-length homeodomain (with the characteristic 60 aminoacid sequence shown in black), or a truncated homeodo-main. Modified from Latchman [33].

330 L.L. OLIGNY

eye in this ectopic site. Such ectopic eyes are de-void of optic nerves, and are therefore nonfunc-tional. In humans, a mutation of PAX-6 causesocular, cerebral, and facial anomalies. Both copiesof PAX-6 need to be expressed to obtain a normalphenotype; if only one copy is expressed, there ishaploinsufficiency, leading to ocular maldevelop-ment. The lack of both copies of PAX-6 results inanophthalmia associated with craniofacial and ce-rebral abnormalities [12,13].

Zinc-finger proteins. The three-dimensionalstructure of these proteins resembles fingers linkedthrough zinc molecules (Fig. 7). Most proteinswithin this family possess two essential domains:(1) a DNA-binding domain consisting of at leasttwo highly conserved zinc fingers, and (2) a ligand-binding domain (e.g., the hormone-binding por-tion of the molecule) that modifies the transcrip-tional activity of the factor.

Among the proteins with zinc-finger domainsare the receptors of the steroid hormone family,thyroxine, retinoic acid, and vitamin D. Differ-ences in the ligand-binding domain enable thesereceptors to bind different hormones and smallvariations in the zinc-finger domain enable them to

bind to different promoters and thus to controldifferent sets of subordinate genes.

Master switch genes

Master switch genes are transcription factor geneswhose proteins control and regulate key events inmorphogenesis through the activation and inhibi-tion of many subordinate genes (Fig. 8). Hence, bydefinition, the activation of a single master switchgene allows the synchronous regulation of a largebattery of subordinate genes necessary for the dif-ferentiation of cells or tissues.MYF-3: AN EXAMPLE OF A MASTER SWITCH GENE. Skel-etal cross-striated muscle cells are the result ofgradual programming. Their undifferentiated an-cestor cells, under the influence of inducing fac-tors, become committed to take the mesodermaldifferentiation. In the embryonic somites, theyconstitute the part known as the myotome. Theirterminal differentiation occurs when the geneMYF-3 (also known as myoblast determininggene 1 [MYO-D1]) becomes expressed. This geneis a master switch gene: any primitive mesenchy-mal cell expressing it becomes a skeletal myo-

Figure 7. Zinc finger transcription factors. To the left,zinc finger proteins in two human receptors: the retinoidX-receptor (A) and the thyroid hormone receptor (B).Each letter corresponds to an amino acid, and the varia-tion in the amino acid sequence within the two fingersprovides specific binding to the subordinate promotersof these transcription factors. Some zinc-finger proteinspossess more than five fingers. C: Zinc-finger protein at-tachment to DNA molecule is shown. The two zinc atomsare in light gray and the protein-zinc bonds in dark gray.The protein interacts with a major groove of the DNAthrough its two fingers, which are helical protein struc-tures similar to those of helix-loop-helix.

Figure 8. Master switch genes. As a general rule, mas-ter switch genes tend to auto-activate their promoters ina positive feedback manner, to ensure a constant state ofactivation in expressing cells and their descendants; thefactors controlling (activating and inhibiting) the masterswitch genes are for the most part unknown. The pro-teins produced by master switch genes can activate orinhibit the transcription of their subordinate genes.


cyte. The MYF-3 protein is a transcription factorthat binds many different promoters specific toskeletal muscular differentiation. Thus, a singlegene is sufficient to activate and inactivate awhole cascade of subordinate genes (Fig. 9). Fur-thermore, MYF-3 recognizes its own promoterand can therefore activate its own transcriptionin a positive feedback loop fashion. Hence, oncea cell activates the transcription of MYF-3, theautoactivation causes this cell and its descen-dants to express MYF-3 forever: this cell line iscommitted to muscular differentiation—fromthen on, these cells can take no differentiationpath other than a myocytic one [14].MYF-5: AN EXAMPLE OF “DOUBLE INSURANCE”. Verte-brates cannot survive without myocytes, and a“double insurance” or genetic redundancy mecha-

nism has evolved to ensure muscular developmentin the event of a loss of function of MYF-3. Asecond gene, called MYF-5, performs roughly thesame functions as MYF-3. Each can substitute forthe other in the event of a mutation (Fig. 10). Inaddition, MYF-3 and MYF-5 bind and cross-acti-vate their promoters, to further ensure double in-surance. At present, four myogenic genes areknown in mammals: MYF-3, MYF-5, MYF-4, andmyogenin. All belong to the same gene family, dis-playing a high degree of sequence similarity. Theyare each able to start the muscle differentiationprogram when injected into fibroblasts. Such re-dundancy is found in many developmentally criti-cal molecular cascades [15].

The genes that control the fundamental as-pects of development do so by controlling a whole

Figure 9. MYF-3: a masterswitch gene. MYF-3 acti-vates the expression ofgenes involved in skeletalmuscle differentiation andinactivates unrelatedgenes.

332 L.L. OLIGNY

battery of subordinate genes. Furthermore, the ex-pression of a selector gene at different moments ofembryonal development may have different ef-fects, as the promoters of subordinate genes arenot all available for modulation at the same time.Therefore, one master switch gene may be used atdifferent times and/or in different regions to per-form different tasks, increasing the efficiency ofthe genome (fewer genes are necessary than if eachfunction required a single-purpose gene).

Genomic imprinting

Genomic imprinting (also referred to as gametic orparental imprinting) is the epigenetic marking of agene based on its parental origin that results inmonoallelic expression. Genomic imprinting differsfrom classical genetics in the sense that the parentalcomplement of imprinted genes are not equivalentwith respect to their expression, despite both parentscontributing equally to the genetic content of theirprogeny. The mechanism of imprinting is complexand not completely understood; however, evidencesuggests that the “imprint mark” is a parental-spe-cific methylation of CpG-rich domains that is estab-lished during gametogenesis. The imprint marks ona gene are erasable in the germline but maintainedduring somatic cell division.

Genomic imprinting plays a critical role inembryogenesis, particularly in the extraembryonictissues, as evidenced by certain aberrations of hu-man pregnancy. The complete hydatidiform molearises from the fertilization of an anuclear eggeither by a haploid sperm (followed by duplicationof the paternal genome) or two haploid sperm (di-andric diploidy). This trophoblastic disease ischaracterized by a completely androgenetic ge-nome and results in reduced or absent fetal growthcoupled with hyperplastic extraembryonic (placen-tal) growth. Similar parent-of-origin effects areseen in human triploids. This demonstrates thatgenes expressed exclusively from one parental ge-nome exist, and abnormal embryonic or gesta-tional sac development results from the loss offunction of the other parent’s monoallelically ex-pressed genes [16–18].

Genomic imprinting is a complex process in-volving an interplay of gene-specific and chromo-somal domain events, including DNA methylation,chromatin compaction, and DNA replication. Im-printed genes often reside in chromosomal regionsthat undergo asynchronous replication, and themeiotic recombination frequencies in these re-gions may differ between the male and female

Figure 10. MYF-3/MYF-5:double insurance. The ef-fects of MYF-3 and MYF-5 inmuscular determination areinterchangeable. Therefore,if one of these fails, theother takes over withoutany developmental conse-quences. (I am indebted toDrs. P. Dias and D. Parhamfor sharing with me theirinsights regarding MYF-3,MYF-4, and MYF-5.)


germ cells. Genes have been identified that encodefor untranslated RNA and antisense RNA that maybe involved in imprint control.

Experimentally it has been shown that a largenumber of imprinted genes are expressed in thebrain and the pituitary. The potential role of im-printed genes in brain development is suggested bythe nonrandom distribution of cells with dupli-cated paternal (androgenetic) or maternal (gyno-genetic) genomes in chimeric brains with normalcells. Androgenetic cells contribute mainly to thehypothalamus and other areas important for pri-mary motivated behavior, while gynogenetic cellspredominantly accumulate within the neocortexand the striatum, areas involved in more complexbrain function. Expression of imprinted genes inthe brain and pituitary could be crucial to mam-malian physiology through central regulation ofreproduction by hormonal priming for develop-ment, lactation, and maternal behavior [18].

Several theories have been proposed for theendogenous function of genomic printing. One hassuggested that genomic imprinting in mammalshas evolved from a conflict of interest between thepaternal and maternal genome in regulating fetalgrowth. Propagation of a paternal line might beensured by formation of a large placenta and fetus.On the other hand, a control of fetal and placentalgrowth could ensure more offspring from themother [16]. An alternative theory for imprintingviews the cytosine methylation involved in imprintregulation as a defense mechanism for the inacti-vation of various parasitic sequences such as trans-posable elements and proviral DNA [16]. Irrespec-tive of the reason for the evolution of genomicimprinting in mammals, the functional conse-quences of genomic imprinting include the inhibi-tion of parthenogenesis and, secondarily, the lossof protection from deleterious effects of recessivemutations in the imprinted regions.

Apoptosis: the physiological cell deathThroughout normal embryogenesis, a large num-ber of cells and tissues are eventually resorbed.Upon proper stimulation, cells initiate the apopto-tic cascade, activating autophagic enzymes, result-ing in cellular suicide. Physiological mechanismsof cell death are used by multicellular organismsduring development and morphogenesis to control

cell number, and as a defensive strategy to removemutated, or damaged, cells. Cell death is requiredfor the normal development of almost all multicel-lular organisms. Cells are commonly produced inexcess, with subsequent removal of those that aresuperfluous. While the apoptosis effector mecha-nisms have been extensively characterized, under-standing of the pathways that signal and controldevelopmental cell death is far from complete.Most of the cells produced during mammalian em-bryonic development undergo physiological celldeath before the end of the perinatal period. Thekey effector components of apoptosis are caspases,a family of cysteine proteases [19].

There are about a dozen mammaliancaspases that exist in cell as inactive zymogens.Once caspases are activated, they cleave a largenumber of proteins within the cell, causing its de-mise and resulting in the morphological changes ofapoptosis. Activation of caspase precursors isachieved by adaptor proteins that bind to them viashared motifs. Apoptosis occurs throughout mam-malian development, from the formation of theinner and outer cell mass in the blastocyst. It isinvolved in the formation of tubes, the separationof the digits, the remodelling of bone, and involu-tion of the mammary glands. Syndactyly of fingersor toes is the consequence of a loss of apoptoticresorption of the interdigital skin webs, but anabnormal control of apoptosis is generally associ-ated with much more severe consequences. Forexample, pachygyria is also thought to be the resultof abnormal apoptosis during the development ofthe brain.

The complexity of the apoptotic process canbe well illustrated on the p53 gene. The p53 proteinis a transcription factor that enhances the rate oftranscription of seven known genes carrying outthe p53-dependent functions in a cell [20]. Whilep53 is not required for a specific event in embryo-genesis, it can halt the cell cycle and/or initiate theapoptotic program when activated by damaged orforeign DNA or by a disturbed cell cycle. In addi-tion to p53 and caspases, many other proteins areinvolved in the apoptotic cascade in postnatal life(Fig. 11), but the molecular agents of apoptosisduring embryonal development remain poorly un-derstood [20,21].

334 L.L. OLIGNY

MORPHOGENESISThe complex form of a developing embryo is theresult of a co-ordination between the drivingforces of morphogenesis and the processes of cellgrowth, proliferation, differentiation, and death.Morphogenesis is responsible for bringing cellpopulations together for new inductive interac-tions and for building complex, three-dimen-sional structures such as hearts, limbs, lungs,and eyes out of simple epithelial sheets and mes-enchymal cell masses.

The genetic pathways underlying cell divi-sion, cell fate determination, and differentiationhave shown themselves to be evolutionarily con-served—a set of rules common to processes thatare used repeatedly in different combinations tomake functional organs. These instructions fallinto two categories. First, there are basic subrou-

tines that define essentially mechanical operationssuch as the packaging of cells into segments, thefolding of epithelial sheets into tubes or cups, andthe outgrowth of buds. Each of these modules uti-lizes sets of genes controlling properties such asdifferential cell adhesion, cell motility, cell–matrixinteractions, and cytoskeletal organization.

The second category determines how thesesubroutines are co-ordinated with cell prolifera-tion and cell fate determination. This co-ordina-tion depends on signaling centers that arise inthe organ primordia or progenitor fields at posi-tions initially determined by the primary embry-onic axes. Each center is a group of cells thatregulates the behavior of surrounding cells byproducing positive and negative intercellular sig-naling molecules. Evidence is beginning to accu-mulate that most of these signaling factors are

Figure 11. Apoptosis: the p53-Rb pathway in postnatallife. The molecular cascades responsible for embryo–fe-tal apoptosis are not known at present; nevertheless,some components of the postnatal cascade shown hereare certainly important in prenatal development. Shownhere are the relationships among a number of onco-genes (gray circles) and tumor suppressor genes (blacksquares) that regulate the ability of cells to initiate DNAsynthesis, the first step in the mitotic process (the so-called G1-S restriction point), the “DNA damage check-point” mediated by p53, and the choice by p53 ofwhether to initiate a G1 arrest (via p21) or apoptosis. Rb

and its two related gene products, p107 and p130, ap-pear to play an important role in p53-mediated G1–Sphase regulation. Note the p53–MDM2 autoregulatoryloop that reverses the checkpoint control and the geneproducts that positively or negatively act on the proba-bility of entering apoptosis. Proliferative cell nuclear an-tigen (PCNA) plays two major roles–first through its acti-vation of DNA polymerase and second as a mediator ofDNA repair. E6 is a protein coded by the human papil-loma virus (HPV) genome, to prevent infected cells fromundergoing apoptosis. Adapted from Levine [20] withpermission from the author and from Elsevier Science.


proteins encoded by a relatively small number ofconserved multigene families, in particular thefibroblast growth factors, bone morphogeneticproteins, epidermal growth factors, hedgehogand wingless [22].

The diverse biological activities of individ-ual ligands are regulated by antagonists, activa-tors, or post-translational modifiers that control,for example, the range over which the proteincan function or its half-life in the environment.In addition, the signaling genes themselves areoften transcriptionally regulated by positiveand/or negative feedback loops. All these factorsultimately affect the size, shape, and pattern of aparticular organ.

SegmentationThe mechanisms responsible for segmentation inDrosophila and humans share certain similarities.These mechanisms are better understood in Dro-sophila, where segmentation occurs in a stepwisefashion. Embryos of Drosophila are a syncytium ofnuclei. Segmentation starts in an anteroposterior(A-P) demarcation: a small group of nuclei local-ized in the anterior-most portion of the embryoproduces the transcription factor bicoid, whichdiffuses toward the posterior pole of the embryo.Similarly, nanos, also a transcription factor, is pro-duced posteriorly and diffuses anteriorly. Thereare thus two concentration gradients in the A-Paxis of the embryo. Subsequently, bicoid activatesthe transcription of hunchback, while nanos inhib-its it, through their respective binding of hunch-back’s enhancers and silencers. It is thus their rel-ative concentrations which precisely regulatehunchback’s expression. The concentration gradi-ent of the these two proteins causes a decreasingconcentration of hunchback along the A-P axis ofthe embryo.

The concentration gradient of hunchbackwithin the embryo controls the expression of thegap family of genes, thus molecularly segmentingthe embryo in distinct domains of gap expression.Each of these gap domains is subsequently seg-mented due to a differential expression of ninepair-rule genes, all of which are homologous tovertebrates’ PAX genes. Once the pair-rule seg-ments are well established, the pair-rule proteinscontrol the expression of the segment-polarity

genes that further segment the embryo along thesame axes as the gap and pair-rule domains. TheDrosophila embryo finally develops an independentsegmentation pattern dictated by the homeoticgene complexes (HOM-C). The interaction be-tween the gap, pair-rule, and hom master switchtranscription factors dictates the differentiationpath of each cell of the embryo [23].

Homeotic genes

Homeotic genes control the basic architecture ofDrosophila development, such as the specificationof the localization of the abdominal segment andof the antennas. The homeotic genes are translatedinto transcription factors called homeoproteins.These proteins have a structure that allows them tobind DNA specifically through a sequence of ap-proximately 60 amino acids. This sequence of 60amino acids is called the homeodomain, and it hasbeen extremely well conserved during evolution.By definition, homeoproteins are the proteins con-taining this sequence, but not all proteins contain-ing a homeodomain have homeotic properties.Over 100 homeoproteins exist.

In Drosophila, some of the homeotic genesare intimately linked on two chromosomes to formtwo homeotic complexes (Fig. 12). The topo-graphic arrangement of HOM genes plays a crucialrole in the anatomic and temporal expression ofthese genes. HOM genes seem to play a role in cellproliferation, as well as in segment identity. Forexample, a mutation of antennapedia causes legsto grow in lieu of antennas in Drosophila.

During evolution, the primordial eight-genehomeotic complex was duplicated and rearranged,so as to form four homeotic sequences on fourdifferent chromosomes in mammals (Fig. 12).Each of these sequences (HOX-A, HOX-B, HOX-C,and HOX-D) contains 9 to 12 homeobox (or HOX)genes; these homeotic complexes share a great ho-mology with their insect homologues and amongstthemselves.

The HOX genes are designated according totheir origin and localization (e.g., HOX-A13, HOX-B9, etc). It must be noted that genes derived fromthe same insect HOM gene (e.g., abd-A with HOX-A9, HOX-B9, HOX-C9, and HOX-D9) all sharegreater homology among themselves than they dowhen compared with other genes of the same com-

336 L.L. OLIGNY

plex (e.g., HOX-A1 vs. HOX-A2). Retinoic acid (RA)

acts as a transcription factor and plays a major role

in the spatial and chronologic expression of HOX

genes; HOX-A1, -B1, -C4 and -D1 require only a

small concentration to become active, whereas the

promoters of HOX-A/B/C/D-13 require larger con-

centrations. The promoters of HOX-A/B/C/D 2 to 13

require increasing concentrations for activation.

Hence, the concentration gradient in retinoic acid

that is known to be present in the A-P and medio-

lateral axes of the embryo may be sufficient to

control the chronologic and topographic expres-

sion of HOX (Fig. 12) [24].

In vertebrates, homeoproteins are detected as

early as the blastocyst stage. As the embryo reaches

the bilaminar stage, it is already “segmented”, with

ventral, dorsal, medial, lateral, right, left, anterior

and posterior poles. Thus, the embryo is seg-

mented, with a unique combination of expressed

homeoproteins at the level of each segment. These

different combinations of homeoproteins control

the expression of specific morphoproteins, whose

role is to control the differentiation of cells ex-

pressing them [23,25].

These morphoproteins in turn control the ex-

pression of subordinate genes, which determine the

differentiation pathway of cells expressing them. Af-

ter embryonal plication, i.e., quite late in develop-

ment, the embryo will become segmented by the four

homeotic complexes. Each cell of the embryo there-

fore has a molecular address, which corresponds to

the transcription factors expressed by that cell and

determines the differentiation path for that cell.

Hence, a heterozygous mutation of HOX-D13 has

been found to cause synpolydactyly affecting the

third and fourth fingers of a boy, whereas his ho-

mozygous mother had more severe foot and hand

lateralizing anomalies affecting the third through the

fifth fingers and toes. Heterozygous mutations of

HOX-A13 are known to cause hypoplasia (failure to

grow in the mediolateral axis) of the first metacar-

pals/metatarsals and first distal phalanges.

Figure 12. The two ho-meotic complexes of Dro-sophila (Antp-C and BX-C)are compared with the fourhuman homeotic com-plexes. In vertebrates, Hox 9to 13 arose from Abd-B.Note that these genes aretranscribed sequentially,starting at the 39-most geneof the complex (HOX-A1/B1/C4/D1), and ending with the59-most genes (HOX-A/B/C/D-13). The retinoic acid con-centration gradient (depict-ed as a grey triangle)probably plays a major rolein the transcription of thesegenes.


Cell motilityCell motility becomes first demonstrated duringgastrulation. It may involve active movements orpassive displacement such as invagination, immi-gration, polar differentiation, and epiboly (a typeof cell movement that involves an epithelial super-ficial layer expansion with spread over the under-lying cells to envelope them). Expansion of theouter layer may be brought about by crawlingmovements supported by cell division and changesin cell adhesiveness. The intensity of cell move-ment increases with growing complexity of mor-phogenesis. The neural crest cells of all vertebratesdisplay extensive migratory activity. Primordialgerm cells migrate from the secondary yolk sacinto primitive gonads. Physical forces, adhesion,and repulsion in addition to electrostatic and ionexchange properties, all closely controlled by pro-teins located on cell membranes, influence cell mo-tility [15].

Cell communication and interactionComplex processes, such as spatially and tempo-rally ordered cell differentiation, directed cellmovement, and shaping of structures composed ofseveral cell types, all require cell-to-cell communi-cation and co-ordinated cell interaction. Manymolecules are used to convey signals. Ions andsmall polar (hydrophilic) molecules, such as Ca21,cAMP, and cGMP, are able to diffuse from cell tocell through gap junctions between cell mem-branes of adjacent cells.

Small molecules with low polarity may di-rectly diffuse across cell membranes. This appliesto molecules such as CO2 and NH3, which are notgenerally considered to serve in communication,as well as to several well-established signal mole-cules. For example, arachidonic acid functions as atranscellular signal molecule. Many metabolites ofarachidonic acid, such as prostaglandins, leukotri-enes, and eicosanoids, are liberated into the extra-cellular spaces and may have the function of spe-cific, locally acting signal molecules. From theextracellular space, such molecules may infiltrateand cross the membrane of neighboring cells, orthey may be picked up by neighboring cells withmembrane-associated receptors.

The significance of low-molecular-weight,non-peptide molecules for embryogenesis is

largely unexplored, because such molecules arefrequently very unstable and are extremely difficultto trace. Larger, lipophilic molecules, such as reti-noic acid, thyroxin, and steroid hormones, arecommonly said to freely cross cell membranes andto bind receptors only within the cytoplasm. How-ever, while their lipophilic nature enables them toinfiltrate membranes, it impedes their detachmentfrom the membrane and their spread into theaqueous cytosol. Therefore, the cytoplasmic recep-tors probably collect lipophilic signal moleculesfrom the cell membrane [15,26].

The counterparts of diffusible signal mole-cules are glycoproteins that act as signal moleculesbut remain anchored in the membrane of the sig-nal-presenting cell. Only adjacent target cells cansense the presence of such a signal exposed on thecell surface. Glycoproteins that enable the mutualrecognition of neighboring cells typically have theadditional function of glue, mediating the physicalcoherence of the cells. They are thus called cell

adhesion molecules (CAMs). The CAMs are dividedinto two large super families. The first consists ofCAMs with an immunoglobulin-like structure (Fig.13), e.g., the cadherin families (including the E-cadherin, the N-cadherin, and the P-cadherin fam-ilies), and the N-CAMs, Ng-CAMs, L-CAMs,A-CAMs, and I-CAMs families. The second super-family is the integrin family, which contains morethan 20 members.

CAMs are localized on cell surfaces and me-diate the adhesion of cells with their neighbors(Fig. 14). Different cell types express differentCAMs [11]. Each type of CAM is attracted by itsidentical type and may attract or repulse othertypes of CAMs. When cells share CAMs that areattracted to one another, these cells are essentiallyattached together. These attraction forces are spe-cific enough to cause the dissociated cells of a frogembryo to reassemble with the ectoblast on thesurface, an intermediate mesoblastic layer, and theendoblast in the center. Some CAMs allow the ex-change of signals between signaling and neighbor-ing subordinate cells, either through CAM–CAMinteractions or through their production of specificextracellular matrix components. When bound to aspecific ligand, some CAMs form dimers, thus ac-tivating their tyrosine kinase function.

338 L.L. OLIGNY

Cadherins are expressed on the cytoplasmicmembrane, at the level of the desmosomes. Theyanchor cells of a same type together. For example,during neurulation, the cells of the neural plateand those of the neural gutter express the samecadherin. Once the gutter has formed the noto-chord, the neural tube, the neural crest, and thesomites, these structures change their cadherin ex-pression; the different cadherins glue cells of thesame type together, in addition to providing an-choring sites for the intracellular actin filaments.Hence, with their ability to glue cells together andto interact with actin, cadherins play a major rolein cell migration and in tissue formation.

In addition to sticking to one another, cellsanchor onto the extracellular matrix through inte-grins at the level of cytoplasmic membranes. Inte-grins can bind to the laminin of basement mem-branes and to other molecules of the extracellularmatrix. When cells are anchored by their integrins,intracellular actin filaments bind the integrins andallow cells to pull themselves and thus move whenactin contracts. CAMs and integrins are an essen-tial part of the cell migration machinery essentialfor morphogenesis [27].

Occasionally, neighbouring cells interact viamembrane-associated enzymes. For example,

membrane-anchored glycosyltransferases may

transfer monosaccharides present in the extracel-

lular fluid onto acceptor molecules in the mem-

brane of adjacent cells. This addition of a sugar

moiety may result in a conformational change in

the acceptor molecule, thus modulating signal

transduction pathways.

Similarly, membrane-anchored polypeptides

can be cleaved off by enzymes exposed on the

exterior surface of neighboring cells. This cleaved

peptide becomes a diffusible, extracellular signal

molecule. For example, the epidermal growth fac-

tor can be split off from a membrane-bound pre-

cursor polypeptide.

Diverse molecules with signaling functions

are released into the extracellular fluid that fills the

spaces between cells. Depending on their biologi-

cal function, the location of their release, or tradi-

tion, such substances are called morphogens, in-

ducers, growth factors, differentiation factors, tissue

hormones or paracrine hormones, modulators, me-

diators, elicitors, or transmitters [28]. The distances

of their spread range from a few nanometers to

several millimeters. Some of these factors may also

be chemoattractants, directing the movement or

growth of cells.

Figure 13. A: Diagram-matic representation ofsome members of the im-munoglobulin (Ig) super-family of adhesion mole-cules. The membrane-bound IgM has two heavychains, each with five do-mains, and two light chainswith two domains. Thesedomains are also used byT-cell receptors, major histo-compatibility complex(MHC) types I and II, andN-cell adhesion molecules(N-CAM). Although theyalso belong to the Ig-super-family, I-CAMs, V-CAMs, L-CAMs, and cadherins arenot shown here. B: N-CAM-specific recognition andbinding of cells. Note thatN-CAM represents a wholefamily of similar molecules,not a given molecule.


If signal molecules are delivered into blood orlymph vessels, they have the potential to be distrib-uted throughout the body and thus to co-ordinatedevelopmental processes over large distances indiverse and remote parts of the body. Such hor-monal signal molecules are well suited to co-ordi-nate extensive developmental processes, such asmetamorphosis and sexual development, but can-not mediate positional information or act as localinducers.

Many macromolecular substances are releasedinto extracellular spaces to fill them and to confercertain physical properties to the tissue. The pro-teins, glycoproteins, and proteoglycans that consti-tute the extracellular matrix serve as filling and hard-ening materials, but they also can serve as signpostsand guides, directing the movement of wanderingcells and stimulating the growth of nerve fibres andblood capillaries. Fibronectin, laminin, and hyal-uronic acid are known components of the extracel-lular matrix having signal function.

Region-specific diversityRegion-specific diversity is also known as embry-

onic induction. It is loosely connected to the

concept of morphogenetic fields, which are areas

whose cells can cooperatively bring about a dis-

tinct structure or set of structures. Morphoge-

netic fields define developmental potencies. In

induction, inducing cells instruct or permit sig-

nal-receiving cells to take a specific developmen-

tal path. Inducing signal molecules are usually

present in minute quantities and for a short pe-

riod of time. There are at least four different

general factors responsible for the induction of

region-specific diversity:

1. The particular quality of the locally acting sub-

stances. Factors may be similar but not iden-

tical in the various body regions; they might

even belong to the same protein family, but the

different members are expressed in a region-

specific pattern. For example, several members

Figure 14. Cell adhe-sion molecules. A, B: Asactin is bound to the cad-herins at the level of thedesmosomes, its contrac-tion causes the apex ofthe sheath of cells to con-strict, as the basementmembrane is not elastic.Such a constriction con-tributes to the formationof the neural groove. C:The cell at the migrationfront sends out projec-tions to feel the extracel-lular membrane for mole-cules for which theirintegrins have an affinity.Once such molecules arefound, they are boundand used as anchors toallow actin to pull the cellin that direction. Adja-cent cells are dragged bythe leading cells throughCAM binding.

340 L.L. OLIGNY

of the hedgehog family are present in verte-brates and are expressed in different spatialpatterns: banded hedgehog is expressedthroughout the neural plate and in the der-matome part of the somites, cephalic hedgehogis expressed in ectodermal and endodermalstructures of the head, and sonic hedgehog isexpressed in the notochord and limb buds.

2. The local concentration of factors. The concen-tration may take the form of a gradient ratherthan being spatially uniform.

3. The locally changing proportions in the mixtureof inducing factors. Developmentally importantgenes have large numbers of silencers and en-hancers controlling their promoter. As with med-ications, mixtures differing in their quantitativecomposition may have different effects.

4. The locally different competences of respond-ing tissues. As a consequence of their priordifferentiation, various recipients of the samesignal molecule may respond differently.

Organ primordiaIn vertebrates, budding invariably involves dy-namic and reciprocal interactions between two cellpopulations—one mesenchymal and the other ep-ithelial. A primary bud is defined as a knob-likecluster of progenitor cells located within distinctboundaries that proliferate and move away fromthe surface of a pre-existing structure. A secondarybud develops on a stalk derived from another bud,while branches arise by bifurcation of a terminalbud [22,29].

Other distinct mechanisms for subdividing abud also exist. For example, a terminal, ampulla-like bud may be cleaved into multiple lobules bythe process of clefting, involving the ingrowth ofmesenchyme and the deposition of extracellularmatrix. In some organs, such as the lung, budding,branching, and clefting each occur at differentstages of development (see below), while in organssuch as the salivary gland, for example, cleftingappears to predominate.

Simple bud formation involves several stages.The first is initiation, in which the boundaries of abud primordium, and the signaling centers withinit, are gradually established at a specific site on theA-P, dorsal–ventral, and medial–lateral axes of the

embryo. The second phase, proximal–distal out-growth, is usually, but not always, associated withincreased cell proliferation, mediated by at leastone source of mitogen such as Sonic hedgehog(Shh) or fibroblast growth factors (Fgfs). Duringthis phase, the extending bud retains its positionalco-ordinates, which are important for later pat-terning events. Finally, there is cessation of out-growth, due in part to the programmed death, dis-placement, or inhibition of distal mitogen-secreting centres. Initiation of a primary budappears to be a dynamic process in which initiallybroad domains of gene expression become gradu-ally restricted.

Bud outgrowth in vertebrates is invariablyassociated with increased cell proliferation medi-ated by distal sources of mitogens such as Fgfs andShh. However, several strategies may have to beemployed to ensure that the enlarging bud ac-quires a specific shape and does not just blow uplike a balloon. The most important of these is theestablishment of a balance between self-enhancingmechanisms that activate proliferation or exten-sion distally and counteracting mechanisms thatspecifically inhibit these activities more proxi-mally. It is likely that the physical dimensions andlocation of the mitogen-signaling centers have aprofound influence on the final shape of a bud-derived structure [29,30].

Two other processes besides cell proliferationmay play a role in determining bud outgrowth andshape. One is the elaboration of physical con-straints such as an inflexible ectodermal casingthat forces cells to translocate distally like tooth-paste through a tube, or constricting collagen bun-dles that promote clefting of large buds. Anotherprocess is cell movement, including convergence,extension, and compaction. There is evidence thatlung epithelial cells translocate toward a distalsource of chemoattractant [22].

An incredible example of migration and epi-genetic self-organization is the development ofneuroblasts and neural connections within thecentral nervous system. The nervous system is byfar the most architecturally complex human organ.Looking at the known facts about its development,one can see how much is missing from our molec-ular understanding of the embryology of the cen-tral nervous system. The beginning of our under-


standing of the given complexity can bedemonstrated through an example of neuronal mi-gration and connecting the neurons with their pe-ripheral targets.

Differentiated neurons no longer divide, andtherefore, the transcription factors that are ex-pressed at their last differentiation division deter-mine the course of their further development. Asexpression of transcription factors is a dynamicprocess over time, the generation of neurons of asame type generally occurs during a limited timeperiod. The neuroblasts are located in the neuraltube containing glial cells that extend from thecenter of the tube to its periphery, like the spokesof a wheel. The germinal neurons can be foundadjacent to the lumen of the tube, but as theyproliferate, the maturing neurons migrate to anincreasingly peripheral position with each newgeneration of neurons; the cells generated first mi-grate the shortest distance, the last ones, the long-est.

The location of a neuron is important as it iscontrolled by expression of HOX genes and of mul-tiple other morphogens, all essential to orchestrateorderly intercellular connections. While maturingand migrating, neurons develop projections calledneurites (the future axons and dendrites). The dis-tal extremity of neurites develops a growth conewith cytoplasmic projections that advance and re-treat, feeling along for CAMs with which they shareaffinities and being repelled by CAMs located inregions where they ought not to go. This chemo-taxis is controlled by integrin–extracellular matrixmolecules and it is this interaction that guides themigration of neurites. Because neurites of a similargroup share the same CAMs, they migrate togetherand stay associated in a single tract without min-gling with other tracts, accounting for the orderlyarchitecture of the peripheral and central nervoussystem [15].

In Drosophila, the Down syndrome cell adhe-sion molecule (Dscam) is known to encode an axonguidance receptor with an extracellular domainthat contains ten immunoglubulin repeats;Dscam’s pre-mRNA can be alternatively splicedinto more than 38,000 different mRNA isoforms.This is 2 to 3 times the number of predicted genesin the entire organism. Each mRNA is translatedinto a distinct receptor with the potential ability to

interact with different molecular guidance cues,directing the growing axon to its proper location[31].

Once a neurite reaches its target (e.g., smoothmuscle cell), it is stimulated by a trophic substancesecreted by this target (e.g., nerve growth factor[NGF]). Without this stimulation, the neuron dies.The target produces enough of this substance toallow the survival of an adequate number of neu-rons; as neurons are produced in excess, this sys-tem allows for the elimination of the surplus neu-rons by apoptosis. At this stage, persisting neuritesestablish nonspecific connections with the largestpossible number of targets [15].

The elimination of nonspecific synapses fol-lows. The synapses that specifically control a targetare not harmed and continue that control, whereasthe nonspecific connections are resorbed.

CONCLUSIONSThis overview of human molecular embryogene-sis focusing on embryonic cell growth and mor-phogenesis may give the reader a false impres-sion of a great depth of understanding andinsight into these processes. In reality, ourknowledge is fragmented and in many areasstrictly confined to the details of molecular em-bryogenesis of Drosophila, earthworm, or mouse.The best illustration of complexities in obtaininghuman data is the scarcity of molecular data formorphogenesis of human placenta. The placentais a simple organ comprised of an inner vascularnetwork covered by an outer epithelium calledtrophoblast. The organ is designed to deliver nu-trients to the embryo and the fetus. A recentreview of genetic insights into trophoblast differ-entiation and placental morphogenesis [32] indi-cates significant advances in our understandingof the regulation of mouse placental develop-ment. In mice, the removal of a gene and obser-vation of the loss of its function to obtain knowl-edge of its function is a well–worked outlaboratory procedure. Follow-up experimenta-tion with mouse embryonic implantation andtrophoblast differentiation can be performed forspatial and temporal patterns of expression ofthis gene during the whole gestation. The situa-tion in humans is different, as gene knockoutexperiments cannot be done and the experimen-

342 L.L. OLIGNY

tation with patterns of expression for any placen-tal gene are confined to in vitro studies and toselected gestational ages. Molecular findings inmouse are difficult to transfer directly to humanplacental morphogenesis, as uteroplacental mor-phogenesis of these two species vary consider-ably. Therefore, although human molecular em-bryology is a rapidly evolving, complex field,there is a long way to go before we can expect topinpoint molecular mechanisms for all aspectsof human normal and abnormal embryogenesis.

A C K N O W L E D G M E N T S

I thank Dr. D.K. Kalousek for her guidance andconsiderable help in rewriting this text from itsoriginal form as a syllabus to my course given atthe March 2000 meeting of the Society for Pediat-ric Pathology. I also thank Dr. Tom Seemayer, myparents and in-laws, my wife Ina Angelidou, andmy children Alexandre (13), Marie-Anne (11), andNicolas (9) for their ongoing support at all levels.

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human molecular embryogenesis: an overview

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