Heart development
Heart development refers to the embryological processes that transform mesodermal progenitor cells into a functional, four-chambered organ during early human gestation, beginning at the end of the second week with gastrulation and culminating in a septated heart capable of coordinated circulation by the eighth week.[1][2] Cardiac progenitor cells arise from the epiblast lateral to the primitive streak, migrating to form bilateral heart fields that fuse midline to create a primitive heart tube around day 21, marking the first organ to exhibit contractile activity in the embryo.[2][1] Subsequent morphogenesis involves rightward looping of the heart tube between days 22 and 28, establishing the basic topological arrangement of future chambers, followed by partitioning via endocardial cushions and septa to divide the single atrium and ventricle, while outflow and inflow tracts align for efficient blood flow.[3][1] This tightly regulated cascade, driven by genetic programs including transcription factors like Nkx2.5 and Gata4, integrates proliferation, differentiation, and hemodynamic forces to yield a mature structure resilient to postnatal demands, with disruptions frequently resulting in congenital anomalies affecting up to 1% of live births.[4][5] Key milestones include the onset of heartbeat by day 22 and completion of septation by week 7, underscoring the heart's rapid evolution from a peristaltic tube to a valved pump.[1][3]
Specification and Initial Formation
Cardiogenic Mesoderm Induction
Cardiogenic mesoderm induction refers to the specification of lateral plate mesoderm cells into progenitors committed to cardiac lineages during early gastrulation in vertebrate embryos. This process occurs in the anterior region of the embryo, where primitive streak-derived mesodermal cells receive inductive cues to express cardiac-specific transcription factors. In mice, a model for mammalian cardiogenesis, this specification begins around embryonic day 6.5-7.5, with cardiogenic progenitors migrating anteriorly to form bilateral heart fields.[6] In humans, analogous events transpire approximately 16-18 days post-fertilization, preceding overt heart tube formation.[4] Induction primarily arises from interactions between overlying ectoderm, underlying endoderm, and nascent mesoderm, with anterior visceral endoderm (AVE) in mice or anterior endoderm in other vertebrates secreting diffusible signals. Experiments in chick and frog embryos demonstrate that anterior endoderm explants can induce cardiac gene expression in non-cardiogenic mesoderm, indicating its instructive role independent of mesoderm-intrinsic programs.[7] Bone morphogenetic protein (BMP) signaling, particularly BMP2 and BMP4 from lateral plate mesoderm and endoderm, is essential for initial mesoderm specification toward cardiogenic fates, activating downstream targets like GATA4 and NKX2.5.[8] Fibroblast growth factor (FGF) pathways, including FGF8, cooperate transiently with BMP to promote proliferation and differentiation of these progenitors, as evidenced by loss-of-function studies in zebrafish and mice where FGF inhibition abolishes cardiac markers.[9] Wnt/β-catenin signaling exhibits biphasic regulation: canonical Wnt promotes primitive mesoderm formation but must be antagonized anteriorly for cardiogenic commitment, via inhibitors like Dkk1, Crescent, and Frzb secreted by endoderm.[10] Overexpression of Wnt antagonists in posterior mesoderm induces ectopic cardiac tissue, underscoring their necessity.[10] Nodal/TGF-β signaling upstream integrates with these, driving mesendoderm formation and Eomesodermin (Eomes) expression, which licenses primitive mesoderm for cardiogenic potential.[6] Mesoderm-specific transcription factor Mesp1 patterns nascent mesoderm into cardiac subsets by restricting alternative fates like somitogenesis, with Mesp1-null mice exhibiting severe cardiac hypoplasia.[11] Core cardiac transcription factors emerge post-induction: NKX2.5, GATA4, and TBX5 form regulatory networks amplified by BMP and FGF, with NKX2.5 mutants in mice showing disrupted mesoderm migration despite initial specification.[12] Id genes, as BMP effectors, inhibit non-cardiac repressors like Tcf3 to facilitate differentiation.[13] These mechanisms are conserved across vertebrates, though human pluripotent stem cell models reveal nuanced differences, such as enhanced reliance on timed Wnt modulation for efficient cardiogenic mesoderm yield.[14] Disruptions in these pathways underlie congenital defects, linking induction failures to conditions like heterotaxy via genes such as SOX17.[15]Endocardial and Myocardial Tube Fusion
During the third week of human embryonic development, approximately days 19 to 22 post-fertilization, bilateral primordia of the endocardium and myocardium converge in the ventral midline to form the primitive heart tube through a process driven by embryonic folding. Cephalocaudal and lateral folding of the embryo positions the paired endocardial tubes, originating from angioblastic cells in the splanchnic layer of lateral plate mesoderm, adjacent to one another.[16][2] These endocardial tubes initially appear as paired endothelial-lined channels within the cardiogenic mesoderm.[17] Fusion initiates in the cranial region near the buccopharyngeal membrane and proceeds caudally, uniting the endocardial tubes into a single conduit by day 22. Concurrently, myocardial precursor cells, also derived from the cardiogenic mesoderm, differentiate into a sleeve of contractile myocardium that envelops the endocardial tube, with an intervening acellular layer of extracellular matrix termed cardiac jelly. This results in a trilayered primitive heart tube: inner endocardium providing endothelial lining, cardiac jelly as a supportive scaffold, and outer myocardium responsible for contractility.[16][18] The myocardium at this stage exhibits peristaltic contractions, initiating blood flow shortly after fusion, around day 23.[2] In avian models such as the chick embryo, equivalent fusion occurs between the 1- to 13-somite stages, starting with ventricular primordia and extending to outflow and atrial regions by 8 to 9 somites, facilitated by the ventral mesocardium which anchors the heart tube to the foregut.[19] Endocardial cells actively contribute to myocardial alignment and fusion; experimental disruptions, such as ablation of endocardial progenitors, prevent proper myocardial tube closure, leading to cardia bifida where unfused bilateral heart primordia persist.[20] Molecular signals, including VEGF and Notch pathways, regulate endocardial-myocardial interactions essential for tube integrity, with defects in these signaling causing fusion failures observed in animal models.[21]Morphogenetic Remodeling
Heart Tube Looping and Asymmetry
The straight heart tube, formed by the fusion of bilateral endocardial primordia around day 21 of human gestation, undergoes looping morphogenesis beginning at approximately day 22-23 (Carnegie stage 10). This process transforms the linear tube into a dextral C-shaped loop, with the cranial portion bending ventrally and to the right, while the caudal portion shifts dorsally and leftward, establishing the bulboventricular configuration.[2][3] Looping is complete by day 28, aligning the future outflow tract cranially, the ventricle caudally and rightward, and the atrium dorsally.[2] Mechanically, looping arises from differential cell proliferation and cytoskeletal rearrangements in the myocardium, particularly at the atrioventricular and bulboventricular junctions, coupled with extracellular matrix remodeling via cardiac jelly. Computational models indicate that non-uniform growth and myocardial trabeculation contribute to the tube's helical twisting, with the rightward bias emerging from asymmetric tension in the dorsal mesocardium before its resorption.[22][23] In chick and mouse models, which recapitulate human looping dynamics, this involves ventral bending followed by rightward torsion, driven by left-right differences in cell shape and motility.[24] Left-right asymmetry in looping is orchestrated by a genetic cascade initiated at the embryonic node, where nodal flow generates asymmetric Nodal signaling on the left lateral plate mesoderm (LPM). This activates left-specific expression of the transcription factor Pitx2, which patterns second heart field progenitors and directs asymmetric morphogenesis of the outflow tract and atria.[25][26] Pitx2-null mice exhibit randomized or reversed looping, underscoring its role in enforcing dextral chirality, while human heterotaxy syndromes often involve mutations in Nodal pathway components, linking disrupted asymmetry to congenital heart defects like dextrocardia.[27][28] The integration of biomechanical forces with Pitx2-mediated gene expression ensures precise chamber positioning, critical for subsequent septation and valve formation.[29]Cardiac Jelly and Cushion Formation
The cardiac jelly is an acellular, gelatinous extracellular matrix secreted by myocardial cells into the space between the endocardium and myocardium of the primitive heart tube during early cardiogenesis, typically forming a thick cuff that provides structural support and facilitates morphogenetic processes.[18] This matrix, rich in glycosaminoglycans, fibronectin, collagen types I and III, and proteoglycans, emerges shortly after fusion of the endocardial tubes around embryonic day 8.5 in mice or equivalent stages in humans (approximately 3-4 weeks post-fertilization), acting as a hydraulic skeleton analogous to mesoglea in jellyfish, which maintains tubular integrity against hemodynamic forces.[30][31][18] Cushion formation initiates during heart tube looping, with localized expansion of the cardiac jelly in the atrioventricular canal (AVC) and outflow tract (OFT), driven by myocardial synthesis of matrix components and signaling molecules that induce endocardial cell activation.[32] In the AVC and OFT, a subset of overlying endocardial cells undergoes epithelial-to-mesenchymal transition (EMT), delaminating, migrating through the jelly, and differentiating into mesenchymal cells that proliferate and populate the swelling cushions; this process is triggered by myocardial-derived signals such as transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) diffused via the jelly matrix.[30][32] The cushions initially function as temporary valves to regulate unidirectional blood flow and prevent backflow during looping, with hyaluronan synthesis contributing to jelly expansion and EMT competence around embryonic days 9-10 in chick models, homologous to human stages at 5-6 weeks.[32][33] These endocardial cushions, mesenchymalized from approximately 10-20% of local endocardial cells, fuse and remodel to form the primitive atrioventricular valves (mitral and tricuspid) from AVC cushions and contribute to semilunar valve primordia and septation in the OFT, with neural crest cell migration into OFT cushions aiding partitioning of the truncus arteriosus.[34] Disruptions in jelly composition or EMT, such as hyaluronan deficiencies, lead to incomplete cushion formation and congenital defects like atrioventricular septal defects, underscoring the jelly's causal role in enabling mesenchymal invasion and structural partitioning essential for four-chambered heart functionality.[32][30]Chamber Specification and Septation
Atrial and Ventricular Patterning
Atrial and ventricular patterning initiates in the nascent linear heart tube following fusion of the endocardial and myocardial layers, establishing distinct myocardial identities along the anterior-posterior axis through graded expression of transcription factors and signaling cues. This process divides the tube into regions fated for the primitive ventricle anteriorly and atrium posteriorly, with subsequent ballooning of the outer curvature forming mature chambers. In mice, chamber-specific gene expression emerges around embryonic day 8.5 (E8.5), while in humans, ventricular chambers are evident by day 32 post-fertilization and a four-chambered structure by week 7.[4][35] Key transcription factors drive this specification. Tbx5 exhibits a posterior-to-anterior gradient, with high levels in the future atrium and left ventricle, promoting atrial septation and left ventricular identity through interactions with Nkx2.5 and Gata4; its mutations, as in Holt-Oram syndrome, disrupt septal formation and cause atrial or ventricular septal defects. Nkx2.5, broadly expressed in cardiac progenitors from E8.5 in mice, cooperates with Hand2 and Mef2c to specify ventricular myocardium and trabeculation, with deficiencies impairing ventricular compaction and conduction. Hand1 and Hand2 further delineate ventricular subtypes, with Hand2 directing right ventricular and outflow tract progenitors and Hand1 influencing left ventricular development; disruptions lead to ventricular hypoplasia or double-outlet right ventricle. Gata4 synergizes with Tbx5 and Nkx2.5 to regulate cardiomyocyte proliferation and chamber boundaries, and its absence results in atrioventricular septal defects. Atrial-specific factors like Pitx2 and Coup-TFII (Nr2f1) reinforce posterior identity, while ventricular repressors such as Tbx2, Tbx3, Hey2, and Irx1-6 suppress atrial genes anteriorly.[4][35][36] Signaling pathways modulate these networks to enforce chamber fates. Retinoic acid (RA), active in the posterior second heart field (SHF) from E9.5-E12.5 in mice, promotes atrial differentiation by activating Nr2f1 and atrial markers like Nppa and Mlc2a, while its inhibition favors ventricular identity marked by Mlc2v and Myh7; excess or deficiency causes atrial or ventricular septal defects. BMP signaling, via Bmp10, drives anterior first heart field (FHF) ventricular trabeculation and upregulates Nkx2.5 through Gata4, with Alk3 receptor mutations yielding thin ventricular walls. Wnt signaling exhibits biphasic regulation, initially promoting SHF proliferation and later inhibiting to enable myocardial differentiation and ventricular ballooning; canonical Wnt/β-catenin restricts atrial expansion. Additional inputs like Notch and TGF-β influence atrioventricular canal boundaries to prevent chamber fusion, while left-right asymmetry cues (e.g., Nodal-Pitx2) align atrial appendages.[4][36][35] These mechanisms ensure functional heterogeneity, with atrial cardiomyocytes expressing Hcn4 and Cx40 for rapid conduction and ventricular cells featuring Cx43 and thicker myocardium for force generation. Disruptions, often from compound heterozygosity (e.g., Tbx5-Nkx2.5), yield congenital defects like atrioventricular canal defects or heterotaxy, highlighting the interplay of FHF (left ventricle, parts of atria) and SHF (right ventricle, atria, outflow) progenitors prepatterned in the primitive streak.[4][36][35]Interatrial and Interventricular Septa
The interatrial septum develops from the end of the fourth gestational week, beginning with the formation of the septum primum as a sickle-shaped crest extending from the roof of the common atrium toward the endocardial cushions.[2] This growth leaves an initial opening known as the ostium primum, which permits blood flow between the developing atria.[2] As the septum primum approaches the cushions around the fifth week, the ostium primum closes upon fusion, while perforations in the upper portion of the septum primum coalesce to form the ostium secundum, ensuring continued right-to-left shunting.[37] [2] Subsequently, the septum secundum arises as a thicker, crescent-shaped invagination of the atrial wall to the right of the septum primum, growing downward but not fully to the cushions, thereby overlapping the ostium secundum and creating the foramen ovale.[37] [2] The persistent lower edge of the septum primum functions as a flap valve over the foramen ovale, which remains patent in utero for oxygenated blood passage from the right to left atrium.[37] Postnatally, elevated left atrial pressure typically seals the foramen ovale, with morphological fusion occurring in approximately two-thirds of individuals within two years.[37] The interventricular septum forms concurrently starting in the fourth week, primarily through the muscular component, which arises from the apposition and merger of the medial walls of the developing ventricles, growing upward from the apex toward the endocardial cushions.[2] [38] This muscular portion constitutes the bulk of the septum, leaving a temporary primary interventricular foramen at its superior aspect.[38] Closure of the foramen occurs via the membranous portion, derived from connective tissue of the endocardial cushions and contributions from the truncoconal ridges, completing septation by the seventh week.[2] [38] The membranous septum is thinner and smaller than the muscular part, separating the left ventricle from both the right ventricle and, inferiorly, the right atrium.[38] Full septation divides the single ventricle into right and left chambers, aligning with the completion of atrioventricular and outflow tract partitioning around days 27 to 37 post-fertilization.[2]Atrioventricular Canal Septation
The atrioventricular (AV) canal, initially a common passageway connecting the primitive atrium and ventricle during early heart tube formation, undergoes septation to establish separate right and left AV junctions, enabling unidirectional blood flow to the tricuspid and mitral valves. This process primarily occurs between the 27th and 37th days of embryonic development through the formation, growth, and fusion of endocardial cushions derived from the cardiac jelly matrix secreted by the myocardium.[2] The cushions consist of mesenchymal tissue arising from endothelial-to-mesenchymal transition (EMT) of endocardial cells in the AV canal region, regulated by signaling molecules such as transforming growth factor-beta 2 (TGF-β2) and bone morphogenetic protein 2 (BMP2), which induce EMT and cushion swelling.[34] Septation begins with the development of superior (dorsal or rostral) and inferior (ventral or caudal) endocardial cushions that protrude into the AV canal lumen, followed by the emergence of paired lateral cushions. The superior and inferior cushions elongate toward each other, fusing in the midline to divide the canal into distinct left and right orifices by approximately the 7th week of gestation.[39] This fusion is complemented by contributions from the dorsal mesenchymal protrusion (DMP), a structure originating from the second heart field that provides additional mesenchyme to bridge the primary atrial septum with the AV cushions, ensuring continuity of the atrial and AV septa.[40] Concurrently, the muscular component of the interventricular septum grows upward from the ventricular floor, meeting the fused cushions to complete the membranous interventricular septum.[41] The lateral cushions, forming later, differentiate into the valvular leaflets of the AV valves, with their mesenchymal cells remodeling into fibrous tissue under the influence of transcription factors like Tbx2 and Tbx3, which maintain AV canal myocardial identity distinct from chamber myocardium.[35] Failure in cushion fusion or EMT, often linked to genetic disruptions such as in CRELD1 or GATA4 genes, results in atrioventricular septal defects (AVSD), underscoring the precision of these cellular interactions.[42] Experimental models in mice demonstrate that targeted inactivation of TGF-β signaling impairs cushion mesenchyme formation, halting septation and confirming the pathway's causal role in AV partitioning.[34]Outflow Tract and Valve Development
Truncus Arteriosus Partitioning
The partitioning of the truncus arteriosus occurs during the fifth week of human embryonic development, following the completion of heart tube looping, and involves the division of the common outflow vessel into the ascending aorta and pulmonary trunk via formation of the aorticopulmonary septum.[2] This process ensures separate systemic and pulmonary circulations, with the septum's spiral orientation aligning the aorta with the left ventricle and the pulmonary trunk with the right ventricle.[34] Endocardial swellings, or truncal cushions, initially form in the truncus arteriosus through epithelial-to-mesenchymal transition (EMT) of endocardial cells, generating mesenchymal ridges: a right-superior cushion and a left-inferior cushion.[2] These cushions migrate toward each other—the right-superior toward the left and the left-inferior toward the right—while elongating and spiraling due to differential growth and cellular proliferation.[2] Fusion of the cushions midway along the truncus establishes the aorticopulmonary septum, which extends proximally to connect with the conal septum and distally to the aortic sac, completing septation by approximately the seventh week.[5] Cardiac neural crest cells (CNCCs), originating from the postotic hindbrain (rhombomeres 6–8), play a critical role by migrating through pharyngeal arches 3, 4, and 6 to populate the truncal cushions starting around embryonic day 10 in mammalian models equivalent to early fifth week in humans.[34] These CNCCs contribute mesenchymal cells that reinforce the cushions, promote ridge fusion, and provide structural integrity to the septum; experimental ablation of CNCCs in chick and mouse models results in failure of septation, leading to persistent truncus arteriosus.[43] CNCC migration and differentiation are guided by semaphorin signaling, such as Sema3C-Plxna2 interactions, with disruptions in genes like Gata6 impairing this process.[34] Additional molecular regulation involves BMP4 and TGFβ signaling for cushion maturation, alongside Notch and Wnt/β-catenin pathways that coordinate CNCC invasion and septal alignment.[34] Tbx1 expression in the secondary heart field supports outflow tract elongation, indirectly facilitating partitioning by providing a scaffold for CNCC integration.[34] This coordinated cellular and molecular interplay ensures precise septation, with the truncal region's mesenchymal ridges distinguishing it from proximal conal septation, which relies more on myocardial contributions.[2]Semilunar and Atrioventricular Valves
The atrioventricular (AV) valves, comprising the tricuspid and mitral valves, originate from endocardial cushions that form in the AV canal around the fifth week of human gestation. These cushions arise as swellings of proteoglycan-rich extracellular matrix beneath the endocardium, populated by mesenchymal cells generated through epithelial-mesenchymal transition (EMT) of endocardial cells, with additional contributions from epicardium-derived cells and minor input from cranial neural crest cells.[44] [45] By the seventh to eighth week, superior and inferior cushions fuse to form the septal leaflets, while lateral cushions develop into the mural leaflets, delineating right and left AV orifices and establishing the fibrous annulus that electrically insulates atrial and ventricular myocardium.[46] [44] Morphogenesis of AV valves involves elongation of cushion-derived leaflets, formation of chordae tendineae from subendocardial mesenchyme, and papillary muscle attachment from myocardial progenitors, completing basic structure by the ninth week.[45] The nonmuscular portions of leaflets derive exclusively from AV cushions, which remodel via cell proliferation, extracellular matrix stratification, and hemodynamic influences, with processes advancing through the twelfth week and refining postnatally into thin, flexible structures resistant to regurgitation.[46] [44] Semilunar valves, including the aortic and pulmonary valves, develop from endocardial cushions in the outflow tract (OFT) or truncus arteriosus, initiating around the fifth to sixth week following initial septation by neural crest-derived ridges.[45] These cushions, formed similarly via EMT of endocardial cells with substantial cranial neural crest cell invasion, undergo excavation and sculpting post-partitioning of the OFT into aorta and pulmonary trunk, yielding three cusps per valve by the sixth to ninth week.[44] Cellular semilunar valves first appear at embryonic stage 17 (approximately six weeks), with mesenchymal cores covered by endothelium that differentiates under shear stress—flat on ventricular surfaces facing high flow and cuboidal on arterial sides.[47] Maturation of semilunar valves proceeds through margin growth via localized proliferation in low-flow zones, accumulation of elastic and collagen fibers correlating with pressure fluctuations, and overall thinning, achieving fibroelastic maturity near birth.[47] [44] Disruptions in cushion remodeling or neural crest migration can yield fused cusps, as observed in congenital anomalies, underscoring the precision of these temporally coordinated events.[45]Conduction System Maturation
Pacemaker Node Formation
The sinoatrial node (SAN), the primary pacemaker of the heart, forms from progenitor cells at the embryonic venous pole, specifically within the right sinus venosus, which derives from Tbx18-positive and Isl1-positive mesodermal precursors of the second heart field.[48] These cells originate in the right lateral plate mesoderm and contribute to the pro-pacemaking region, distinct from the primary myocardium of the heart tube.[49] In mammalian embryos, including humans, this process coincides with the incorporation of the sinus venosus into the developing right atrium, establishing the SAN at the junction of the superior vena cava and crista terminalis.[50] Morphological development begins with the specification of pacemaker precursors, followed by proliferation and differentiation. In mouse models, SAN progenitors emerge around embryonic day 8.5 (E8.5), with the initial Tbx3-positive primordium appearing in Hcn4-positive sinus venosus myocardium by E9.5; the node becomes discernible by E10.5–E11.5 through recruitment of mesenchymal cells and maturation by E13.5.[48] [50] In human embryos, the SAN primordium manifests as a myocardial cuff with a tail-like extension at the right atrium–right common cardinal vein junction by Carnegie stage 13 (approximately 32 days post-fertilization, or 4–5 weeks gestation), aligning with the onset of coordinated heartbeat around day 22–23.[3] [51] Early electrical pacemaking precedes overt morphology, initiating in the posterior heart tube before shifting to the SAN region as venous structures remodel.[48] Molecular regulation involves a network of transcription factors that specify and maintain pacemaker identity via mutual repression of chamber myocardial genes. Tbx18 drives initial formation of the SAN head by differentiating mesenchymal precursors into nodal myocardium, while Tbx3 sustains this fate by suppressing atrial markers like Cx40 and Nppa.[52] [50] Shox2 promotes pacemaker gene expression (e.g., Hcn4) by inhibiting Nkx2-5, which otherwise drives working myocardium differentiation; disruption of this balance, as in Shox2-null mice, impairs SAN development.[48] Canonical Wnt signaling initiates precursor specification at the venous pole, with later restriction to the right side influenced by left-right asymmetry cues like Pitx2c, whose absence leads to bilateral SAN formation.[49] [53] These mechanisms ensure localized automaticity, with SAN cells exhibiting slower conduction and distinct ion channel profiles (e.g., elevated HCN4 for hyperpolarization-activated currents) compared to surrounding atrium.[48] ![Embryo at 5 weeks 5 days with heartbeat - annotated.gif][center]This annotated image depicts an embryo at approximately 5 weeks gestation, when early pacemaker activity initiates visible cardiac contractions, reflecting the functional onset tied to SAN precursor specification.[3]