Zac1 is expressed in progenitor/stem cells of the neuroectoderm and mesoderm during embryogenesis: Differential phenotype of the Zac1-expressing cells during development



Zac1, a new zinc-finger protein that regulates both apoptosis and cell cycle arrest, is abundantly expressed in many neuroepithelia during early brain development. In the present work, we study the expression of Zac1 during early embryogenesis and we determine the cellular phenotype of the Zac1-expressing cells throughout development. Our results show that Zac1 is expressed in the progenitor/stem cells of several tissues (nervous system, skeleton, and skeletal muscle), because they colocalize with several progenitor/stem markers (Nestin, glial fibrillary acidic protein, FORSE-1, proliferating cell nuclear antigen, and bromodeoxyuridine). In postnatal development, Zac1 is expressed in all phases of the life cycle of the chondrocytes (from proliferation to apoptosis), in some limbic γ-aminobutyric acid-ergic neuronal subpopulations, and during developmental myofibers. Therefore, the intense expression of Zac1 in the progenitor/stem cells of different cellular lineages during the proliferative cycle, before differentiation into postmitotic cells, suggests that Zac1 plays an important role in the control of cell fate during neurogenesis, chondrogenesis, and myogenesis. Developmental Dynamics 233:667–679, 2005. © 2005 Wiley-Liss, Inc.


Neurogenesis, the processes by which neural cells are generated, occurs through the production of postmitotic neurons and glial-like cells (oligodendrocytes and astrocytes) from neuroepithelial stem cells localized in the ventricular zone of the neural tube by progressive steps of cell division, cell cycle arrest, differentiation, and migration, as well as the natural developmental death of the neural precursors (Roth and D'Sa, 2001; Moskowitz and Lo, 2003). Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors, which control the correct positional identity of the neural cells from the stem/progenitor cells (Johnson et al., 1990; Akazawa et al., 1995; Ben-Arie et al., 1996; Ma et al., 1996; Edenfeld et al., 2002). Several transcription factor expressions contribute to a series of genetic and cellular processes that eventually produce a fully differentiated brain. Zac1, a new zinc-finger protein, is known to be capable of inducing G1 cell cycle arrest and apoptosis, and Zac1 inhibits tumor cell proliferation in vitro and in vivo (Spengler et al., 1997). Thus, Zac1, together with p53, has the singular capacity to simultaneously control these two fundamental cellular mechanisms (Bates and Vousden, 1996; Spengler et al., 1997). The gene encoding Zac1 is located in human and mouse chromosomal regions that are maternally imprinted (Piras et al., 2000; Kamiya et al., 2000; Smith et al., 2002). Zac1 was found to induce expression of the PACAP type 1 receptor in multiple transfected cell lines (Hoffmann et al., 1998). This receptor is the most potent known insulin secretagog and an important mediator of autocrine control of insulin secretion in the pancreatic islet (Yada et al, 1998; Filipsson et al., 1999). Zac1 is mainly expressed in the brain and pituitary gland (Spengler et al., 1997; Valente and Auladell, 2001). In early mouse brain development, Zac1 is strongly expressed in several neuroepithelia, and in adulthood, Zac1 is moderately expressed in some limbic structures, such as hypothalamus, amygdala, hippocampus, and olfactory bulb (Valente and Auladell, 2001). Nevertheless, their biological role in neural development and in mature brain remains uncertain. Furthermore, Zac1 and hZAC are highly expressed in somites and limbs during early embryonic stages (Piras et al., 2000; Valente and Auladell, 2001; Tsuda et al., 2004), as well as in cartilage primordium sites (Valente and Auladell, 2001; Tsuda et al., 2004) and skeletal muscle (Arima et al., 2000; Ma et al., 2004). In adult tissues, hZAC (human homolog of Zac1 mouse gene) and Zac1 are expressed to a low extent in skeletal muscle and bone marrow (Varrault et al., 1998; Piras et al., 2000). The development of skeleton (bone and cartilage) and skeletal muscle in vertebrates begins during early embryonic stages, and their mesodermal origin is interrelated with the ectodermal origin of the nervous system (Bailey et al., 2001; Yang and Karsenty, 2002). Some transcription factors are predominantly expressed in the early phases of the skeletal and skeletal muscle formation, and their functions are restricted to controlling their cell proliferation and differentiation (Yang and Karsenty, 2002). The cartilage and bone cells and the majority of skeletal muscle cells arise from the somites, which are situated adjacent to the neural tube and notochord and respond to signals from the notochord (Bailey et al., 2001). In the present study, our aim was to report a detailed analysis of Zac1 expression during early embryonic stages of development and to characterize Zac1-expressing cell populations during development, by immunohistochemistry and in situ hybridization techniques.


Zac1 Expression in Neural Cells

Embryonic stages.

At E12–E14, strong Zac1 expression was detected within the germinal matrices and weak detectable signals were found in the differentiating fields (Figs. 1, 2; Table 1). In the nervous system, strong expression was observed within active proliferating fields, such as in the neural tube and neural layer of retina, as well as in several neuroepithelia (telencephalic vesicles, third and fourth ventricles, and optic recess). Between E12 and E16, in the innermost cell layer surrounding the lumen of the neural tube and in the ventricular zone of telencephalic vesicles, some Zac1-expressing cells were colocalized with Nestin (Fig. 3B), which recognized an intermediate filament protein (class type VI) expressed in some stem/progenitor cells in the most primitive neuroepithelium, or with glial fibrillary acidic protein (GFAP), which reacted with the class III intermediate filament in both undifferentiating and differentiating astroglia, as well as in stem/progenitor neural cells (Doetsch et al., 1999; Laywell et al., 2000; Alvarez-Buylla et al., 2001; Fig. 3I; Table 2). In addition, many ventricular and subventricular cells that expressed Zac1 gene were colocalized with the proliferative marker proliferating cell nuclear antigen (PCNA), which begins to accumulate during the G1 phase of the cell cycle, becomes most abundant during the S phase, and declines during the G2/M phase (Kurki et al., 1988; Fig. 3F; Table 2). Moreover, some Zac1-expressing cells were colocalized with FORSE-1 within of the ventricular zone and neural tube (Table 2). FORSE-1, an antibody that labels regionally restricted subpopulations of progenitor cells in the embryonic central nervous system and that recognizes the Lewis-X (LeX) carbohydrate epitope, shares expression boundaries with neural regulatory genes and may be involved in patterning of the neural tube by creating domains of differential cell adhesion (Allendoerfer et al., 1999).

Figure 1.

Expression of Zac1 gene in whole mouse embryos at embryonic day (E) 12 (A–E, in toto), E12.5 (F–M, in sections), and E14 (N–U, in sections) by in situ hybridizations. A–E: At E12, intense expression is observed in telencephalic vesicles (tv in A), neural retina (nr in A), the vascular system of the hindbrain (black arrows in A), lateral dermomyotomes (white arrowheads in B), the ventral part of the somites and sclerotome (white arrows in B,C), the cartilage primordium of humerus (hmc in D), and the cartilage condensation of the digital zone of the forelimb (cc in E), as well as the vascular cells that surround the cartilaginous digits of the interdigital zone (open arrows in E). F–M: At E12.5, intense expression is detected in the inner (neural) layer of retina (nlr in F), extrinsic ocular muscle (eom in F), optic recess (or in F), neopallial cortex in the region of the future olfactory lobe (nc in F), ventricular zone of the telencephalic vesicles (vz in G), vascular cells of the interdigital zone (idiz, open arrows in H), and humeral (hmc in H,K and double asterisk in J), radial (rmc in H), phalangeal (double asterisk in H), metacarpal (mc in H) and carpal (single asterisk in H) mesenchymal condensations, as well as in the dorsal premuscle mesenchymal condensation (dmc in H, solid arrows in K and single asterisk in J), cartilage primordium of body vertebrae (cpbv in L), and mantle region of spinal cord in lumbosacral region (mrsc in M). In addition, moderate expression was observed in the neural tube (nt in I,K), in the notochord (n in K) and in the entrance of esophagus (eop in L). N–U: At E14, intense expression is observed in the neural tube (nt in N,P,Q,S,U), notochord (n in N,S), proliferating muscle cells (mpc in N,Q,R,S), muscle cells surrounding the ribs (mr in N), muscle cells that surround the femur (asterisk in N), radial nerve (rn in N), cartilage primordium of femur (cpf in N), cartilage primordium of ribs (cpr in N,P,U), cartilage primordium of neural arch (cpa in N), masseter muscle (mm in O), Meckel's cartilage (mc in O), cartilage primordium of acromion of left scapula (cpas in O), primordium of lower molar tooth (pmt in O), dorsal surface of tongue primordium (dst in O), tooth primordium (tp in O,T), primordium of shoulder joint (psj in Q and R), primordium of joint between tubercle of rib and neural arch of its own vertebra (pj in Q), muscle cells of the limb (mcl in R), cartilage primordium of humerus (cph in R), as well as in the hypothalamus (h in O,T), mamillothalamic tract (mta in O,T), ventricular zone of the telencephalic vesicles (vztv in O,T), neuroepithelium of the third ventricle (nIIIv in O,T), and the plexus choroids (pch in P). In addition, moderate expression is detected in the cartilage condensation primordium of sacral vertebral body (centrum, ccs in U) and aorta area (aa in N), whereas weak expression is observed in the esophagus (o in N), metanephros (definitive kidney, mn in S), and the muscle cells that surround the ribs (mcr in U).

Figure 2.

Immunolocalization of Zac1 protein in the development of the nervous system. A,C: Some migrating Zac1-expressing cells (black cells) are leaving neural tube (nt) region at embryonic day (E) 12. B,D: Zac1-expressing cells in the germinative layers (ventricular, vz, and subventricular zone, svz) of the telencephalic vesicles (B) and in the hypothalamic area (h in D) at embryonic E14. E–G: At E16, many Zac1-expressing cells are found in the ventricular (vz) and subventricular (svz) zones of the lateral ventricles (LV in E) and third ventricle (IIIv in F). G: In addition, in the spinal cord (sc), there is a clear layer with Zac1-expressing cells. Zac1-expressing cells are in black in A–D, and in brown in E–G.

Table 1. Zac1 Expression in Early Embryonic Tissues
Anatomic areaE10E12.5E14
  1. +/−, weak; +, moderate; ++, strong; +++, intense.

Aorta region +/−+
Body wall (thoracic) overlying pericardial cavity+++/−
Cartilage condensation being primordium of sacral vertebral body ++
Cartilage primordium of acromion of left scapula +++
Cartilage primordium of body vertebras +++
Cartilage primordium of femur ++++++
Cartilage primordium of humerus ++++++
Cartilage primordium of neural arch ++
Cartilage primordium of phalangeal bone and metacarpal bone +++
Cartilage primordium of pubic bone +++
Cartilage primordium of radius +++
Cartilage primordium of ribs ++++
Cartilage primordium of thoracic vertebral body +++++
Caudal part of medulla oblongata ++++
Conective tissue of the spinal cord +/−+
Choroid plexus +++
Diencephalon (dorsal and ventral thalamus) ++++
Diencephalon (hypothalamus) ++++++
Digital interzone  +/−
Dorsal surface of tongue +++++
Ependimal layer  ++
Epithalamus +++++
Epithelium of the Rathke's pouch/lumen of anterior lobe of pituitary++++++
Extrinsic Ocular Muscle +/−+
Forelimb bud++ 
Infundibular recess/Infundibulum (future pars nervosa)++++++
Inner (neural) layer of retina +++
Mantle region of spinal cord in lumbosacral region++++++++
Masseter muscle +++
Maxillary bone +/−+
Meckel's cartilage +++++
Metanephros (definitive kidney)  +/−
Middle region of clavicle, with early evidence of ossification  ++
Nasal cartilage +/−+
Neopallial cortex in the region of the future olfactory lobe +++
Neural tube+++++++
Neuroepithelium of forebrain region (telencephalic vesicles)++++++++
Neuroepithelium of hindbrain (aqueduct/fourth ventricle)+/−++
Neuroepithelium of midbrain region (third ventricle)+++++
Oesophageal region of foregut+/−+ 
Oesophagus  +/−
Olfactory epithelium +++
Olfactory placode+/−  
Optic nerve  +
Optic recess ++
Optic vesicle/optic stalk+/−  
Ossification within cartilage primordium of rib  +++
Ossification within cartilage primordium of the humerus  +++
Primordium of follicle of vibrissa  +
Primordium of lower molar tooth  +++
Primordium of pancreas  +
Radial nerve  +
Region of optic chiasma +++
Segmental inter-zone, future localization of intervertebral disc  +
Skeletal muscle of the limbs +++++
Tongue +++++
Tooth primordium  +++
Umbilical vein+++++
Figure 3.

Expression of Zac1 in the neural progenitors of the germinative layers during mouse embryonic and early postnatal development. A: In newborn mice, as in embryonic mice, the Zac1 gene is expressed in the olfactory bulb (ob) and along the rostral migratory stream pathway (open arrows) from the subventricular zone (svz). In addition, some Zac1-expressing cells are found transitorily in the cingular cortex (cg). B–E: Some progenitor/stem cells in the ventricular zone (vz) coexpress the Zac1 gene (in blue) and Nestin protein (in brown; solid arrows in B,C,E), as well as the Nestin gene (in blue) and Zac1 protein (in brown; solid arrows in D), during high mitotic phase of this germinative layer. F–H: In the embryonic and postnatal stages, Zac1-expressing cells (in blue) are colocalized with PCNA (in brown) in the vz and svz of the lateral ventricles, as well as (solid arrows in F,G) in many cells that leave the vz and start a migratory route along the corpus callosum (cc; solid arrows in H). I–K: Progenitor cells express GFAP immunolabel (in brown) in the neural germinative layers. Thus, in embryo (E16) and newborn mice (postnatal day [P] 0) the Zac1-expressing cells (in blue) in the vz are colocalized with GFAP (arrows in I and K). K: In the subsequent postnatal stages (P3), a noticeable decrease in the colocalization between Zac1 and GFAP is observed (see the arrows in the neuroepithelium of the aqueduct of Sylvius, Aq). L,M: In the last stages of embryonic development, some Zac1-expressing cells (in blue color) among the subventricular cells differentiate and start to express PSA-NCAM (in brown; L). M: However, this coexpression is transitory, and at P12, no detectable colocalization is found in vz. N: At E18, the majority of Zac1-expressing cells (in blue) are in the vz and only some of them are in the svz, where they express neuronal markers such as NeuN (in brown). However, when these Zac1-NeuN–expressing cells leave the svz and migrate to the deeper cortical layer (dcl), the expression of Zac1 is completely down-regulated.

Table 2. Cellular Phenotype of Zac1-expressing Cells During Development
First MarkerSecond MarkerDevelopmental ColocalizationAnatomic areas
  1. −, none; +/−, few; +, some; ++, many; and +++, all Zac1-expressing cells are colocalized.

Zac1Nestin++++++/−+/−VZ of several ventricular neuroepithelia; skeletal muscle; follicle of vibrissa
NestinZac1  +++/−+/−+/−VZ of several ventricular neuroepithelia; skeletal muscle; follicle of vibrissa
EGFR1Zac1  ++++/−+/−VZ of several ventricular neuroepithelia; callosum corpus
Zac1FORSE-1++++/−+/− VZ of several ventricular neuroepithelia; skeletal muscle
PCNAZac1++++++++++/−VZ of several ventricular neuroepithelia; chondrocytes; perichondrium; follicle of vibrissa; vascular bone cells; neural layer of retina; skeletal muscle; nasal epithelium; callosum corpus
BrdUZac1 +++++++++/−VZ of several ventricular neuroepithelia; chondrocytes; perichondrium; follicle of vibrissa; vascular bone cells; neural layer of retina; skeletal muscle; nasal epithelium; callosum corpus
ssDNA-ApostainZac1+/−++++/−VZ of lateral ventricles; vascular cells of interdigital zone; postmitotic chondrocytes
DNA fragmentZac1+/−++++/−VZ of lateral ventricles; vascular cells of interdigital zone; postmitotic chondrocytes
Zac1GFAP++++++++/−+/−VZ of several ventricular neuroepithelia
Zac1PSA-NCAM +/−+++/−SVZ of several ventricles; RMS; thalamus
Zac1Vimentin+++/−+/−+/− SVZ of several ventricles
Zac1B-tubulin+/−+/−++SVZ of several ventricles; RMS; hypothalamic and amygdaloid nuclei; olfactory bulb
Zac1Netrin-1+/−++/−  hypothalamus; thalamus; skeletal muscle
Zac1NeuN +/−+++++++in all Zac1 positive areas, excepted in VZ
GAD65Zac1 +/−++++++in all Zac1 positive areas, excepted in VZ, SVZ and brain stem
Zac1Calretinin+++++/−olfactory bulb; marginal cortical layer; enthorinal cortex; hypothalamus
Zac1Calbindin++++++++++in all Zac1 positive areas, excepted in VZ
Zac1Parvalbumin+++++amygdala; CA3 posterior hippocampal regio; hypothalamus
Zac1Somatostatin +/−+/−++hypothalamus; amygdala; CA3 posterior hippocampal regio
NPYZac1 +/−+++++++hypothalamus; amygdala; CA3 posterior hippocampal regio
Zac1TH   +/−+++/−hypothalamus; thalamus; Zona incerta; brain stem; spinal cord
Zac1LHRH    +/−++/−thalamus; olfactory tract; medial eminence
Zac1CFR    ++/−medial eminence; amygdala
Zac1GluR2/3    +/−++/−deeper cortical layers; CA3 posterior

With prosencephalon and mesencephalon development, variable levels of Zac1 gene expression were found in the presumptive olfactory bulb, thalamus, hypothalamus, and amygdaloid area (limbic areas), where a few of these cells were colocalized with β-tubulin (C-terminus of the beta-III isoform of tubulin, which is known to be a specific marker of immature neurons; Tables 1, 2). Furthermore, some Zac1-expressing cells that leave the third ventricle present a migratory cellular route toward the future mamillothalamic tract and dorsomedial and lateral hypothalamic nuclei (Valente and Auladell, 2001). Colocalization studies (Table 2) have demonstrated that these Zac1-expressing cells are positively labeled to the calcium-binding protein calbindin D-28k or calretinin, as well as to Netrin-1 (Fig. 4A), which is a protein implicated in the chemotropic and outgrowth-promoting activities of plate cells to guide commissural axons toward the ventral midline and regulate axon pathway formation and neuronal position during hypothalamic development (Deiner and Sretavan, 1999). At the same time, only very few Zac1-expressing cells are colocalized with NeuN (nuclear neuron-specific protein, which is present in mature neuronal cells of the central nervous system [CNS] and peripheral nervous system; Table 2). Therefore, the Zac1-expressing cells that begin to express neuronal markers (calbindin and calretinin) are not completely differentiated and for this reason they display a much lower reactivity for NeuN.

Figure 4.

Characterization of Zac1-expressing cells in the hypothalamic area. A: At embryonic day (E) 14, the Zac1-expressing cells (in blue) surround the third ventricle starting a migratory route toward hypothalamic region and during this early process they coexpress Netrin-1 (in brown). B,C: All the Zac1-expressing cells that leave the third ventricle are colocalized with calbindin (in brown; B), and this coexpression is maintained throughout adulthood in the hypothalamic (h) and amygdaloid areas (C). D–H: In contrast, only a few Zac1-expressing cells (in blue) of this migratory route in the hypothalamic area (h) are colocalized with calretinin (solid arrows in D–F) and parvalbumin (solid arrows in G,H). I,J: During the establishment of the hypothalamic nuclei between E18 and P5, the neuropeptide Y (NPY) -expressing cells (in blue; I) and GAD65-expressing cells (in blue; J) are colocalized with most of the Zac1 protein cells (in brown). K: In the postnatal stages, some Zac1-expressing cell subpopulations (in blue) are colocalized with Somatostatin (in brown), concretely in the CA posterior region of hippocampus, hypothalamus, and amygdala. L: At postnatal day (P) 9, Zac1 protein is detected in many neuronal cells and axonal projections of the arcuate nucleus and eminence media, respectively. M–O: Many Zac1-expressing cells (in blue) in arcuate nuclei (arc) send their terminations to the eminence media (em) at P9, where they are colocalized with CFR (solid arrows in N). At P3, the Zac1 projections are not actively mature, and the colocalization with CFR (M) and LHRH (O) is much lower.

In subsequent stages (embryonic day [E] 16–E18), there is an increase in Zac1 gene expression in the subventricular zone and, consequently, the number of Zac1-expressing cells that leave this area and begin their differentiating process, especially in the third ventricle. Thus, in the outer cell layer of the subventricular zone of the neuroepithelia, we observed some Zac1-expressing cells that colocalized with differentiating neuronal markers (Table 2), such as PSA-NCAM (polysialic acid neural cell adhesion molecule recognizes the adhesion of neuronal cells; Fig. 3L) and β-tubulin. However, the number of Zac1-expressing cells that colocalized with NeuN was still low (Fig. 4N; Table 2) and was restricted to the amygdaloid and hypothalamic nuclei. At E16, in the limbic areas, some Zac1-expressing cells immunoreacted with several calcium-binding protein markers, such as calretinin, parvalbumin, and calbindin (Figs. 4B,E–H; Table 2), and with the interneuron markers (Table 2), such as the glutamic acid decarboxylase (GAD65), responsible for the conversion of glutamic acid to gamma-aminobutyric acid (GABA), and the neuropeptide Y (NPY). In addition, some cells of the spinal cord expressed GAD65 and tyrosine hydroxylase (TH), which is a catecholinergic marker, and as determined by double-stain techniques, these cells are colocalized with Zac1 protein.

At E18, very few Zac1-expressing cells are colocalized with somatostatin (interneuron subpopulations) in the hypothalamic and amygdaloid nuclei and with TH in the brainstem and spinal cord (Table 2). Nevertheless, the coexpression of Zac1 with TH or somatostatin was more evident during postnatal development (Fig. 4K; Table 2).

Therefore, in the embryonic stages, many cells with high mitotic activity express the Zac1 gene before entering into a differentiation process, which suggests that Zac1 could regulate the proliferation/mitotic phase of the neurogenesis process, probably by the regulation of cell cycle arrest (Spengler et al., 1997). Zac1 is a transcription factor capable of inducing cell cycle arrest in the G1 phase and, by an independent pathway, of inducing apoptosis (Spengler et al., 1997). Recent studies have shown high rates of apoptosis in the neural progenitors during embryogenesis (D'Sa-Eipper and Roth, 2000; D'Sa-Eipper et al., 2001; Zaidi et al., 2001). In this way, we tried to colocalize the Zac1-ventricular cells with apoptotic markers (like ssDNA-F7-26-Apostain or DNA fragmentation), and we observed that the number of Zac1-ventricular cells that die by apoptosis during the proliferation of the neural progenitors is very low (data not shown). Curiously, these apoptotic Zac1-ventricular cells only appear in the lateral ventricles, whereas they are nonexistent in the third and fourth ventricles. Consequently, Zac1 might also be implicated in the apoptotic mechanisms that occur in restricted neural progenitor subpopulations. However, the high expression of Zac1 gene in nonapoptotic neural progenitors of the ventricular zone and their down-expression in differentiated neural cells reinforces the hypothesis that Zac1 might be more involved in the cell cycle arrest of the neural progenitors than in the apoptotic process of these progenitors.

Proliferative areas in postnatal stages.

In newborn animals (at postnatal day [P] 0/P1), a general decrease in the Zac1-expressing progenitor cells was observed, although a restricted Zac1-expressing progenitor pool is maintained within of the ventricular zone and in the rostral migratory stream (RMS) of the olfactory system (Fig. 3A; Table 2). Thus, a very fine line of ventricular cells expressed Zac1 gene and colocalized with EGFR-1, Nestin, GFAP, 5′-bromo-2′-deoxyuridine (BrdU), or PCNA (Fig. 3G,J; Table 2). However, when these progenitor cells begin a differentiation process along the RMS, the Zac1 gene is progressively down-regulated and the Zac1-expressing cells are colocalized with PSA-NCAM and β-tubulin. In the olfactory bulb, The Zac1-expressing cells are completely differentiated and located in the several olfactory layers, mainly in the mitral and granular layers. The expression pattern of the Zac1 gene in the RMS and olfactory system was maintained in the adult brain, although at lower levels (data not shown).

Postnatal Zac1-interneuron subpopulations.

The calcium-binding proteins parvalbumin, calretinin, and calbindin D-28k are markers of different classes of GABAergic interneurons and display different functions (Yan et al., 1995). However, calbindin is not an exclusive marker of interneurons, because it is also in certain glutamatergic neuron populations, such as in the granular cells of the dentate gyrus (Baimbridge, 1992). At P0–P3, many Zac1-expressing cells leave the third ventricle and acquire a differentiation character, since they are labeled positively for neuronal markers (β-tubulin or NeuN) and negatively for macroglial markers (GFAP or Ng2, which is an immature oligodendrocyte marker). Many of these cells in the limbic areas (hypothalamic, amygdaloid, olfactory bulb, and hippocampus) were labeled for calbindin (Fig. 4C; Table 2), whereas some of them in the olfactory bulb were labeled for calretinin (Table 2). In addition, some Zac1-expressing cells in the hypothalamus and amygdala were labeled for parvalbumin (Table 2). In the subsequent stages, we observed a decline in the number of Zac1/calretinin-expressing cells; whereas a noticeable increase in the number of the Zac1/calbindin-expressing cells is found in the limbic areas. In the adult, all Zac1-expressing cells also expressed calbindin protein. In addition, some cortical cells in the cingular area expressed the Zac1 gene transiently during the first postnatal week. These cells also displayed immunoreactivity to the calbindin protein. Moreover, the Zac1/parvalbumin-expressing cells seemed to be more stable throughout postnatal development (Table 2).

In neonates, some Zac1-expressing cells are colocalized with NPY, GAD65, and somatostatin, concretely in the limbic areas (Fig. 4I,J; Table 2); however, it is during postnatal stages that these cell subpopulations represent the majority of Zac1-expressing cells. GAD65 are present in almost all GABAergic neurons (Soghomonian and Martin, 1998). GAD is the biosynthetic enzyme for GABA, the major inhibitory neurotransmitter in the CNS and is strongly expressed in brain development (Lauder et al., 1986; Van Eden et al., 1989). The high cellular levels of both GAD and GABA during early embryogenesis suggests a signalling role during development (Katarova et al., 2000), whereas in mature neural circuits they have a predominant neuronal inhibitory role (Fonnum and Storm-Mathisen, 1969). During embryogenesis, Zac1 gene is expressed in ventricular progenitor/stem cells. With the maturation of the nervous system, these Zac1-expressing cells begin a differentiating process, in which they start to express GAD65 until adulthood. Therefore, all these data together show that Zac1-expressing cells are mainly GABAergic neurons and suggest that Zac1 may be involved in the neuronal differentiation of these GABAergic neuron subpopulations within of the limbic system.

In addition, a few Zac1-expressing cells in the hypothalamic and brainstem areas coexpress TH (Table 2). Colocalization studies confirm the heterogeneity of the Zac1-expressing interneurons, which are nonoverlapping: GABAergic neurons colocalize with somatostatin, NPY, or calcium-binding proteins in the limbic areas; catecholinergic neurons express TH in the brainstem and zona incerta, as well as in the spinal cord. These catecholinergic neurons are probably dopaminergic neurons, because the Zac1 gene is dynamically regulated after in vivo induction of D1 and D2 dopaminergic receptors by the administration of selective agonists and antagonists (Valente and Auladell, unpublished results). In this way, catecholinergic neurons are implicated in the regulation of hormone release in the pituitary gland (Gonzalez et al., 1989; Dorton, 2000). The Zac1 gene is intensely expressed in arcuate nucleus (Valente and Auladell, 2001), which is responsible for the production and release of many hormonal factors in the pituitary gland by the axonal pathway that crosses the eminence media (Szentagothai, 1969). Previous reports (Pagotto et al., 1999, 2000) showed a coexpression of ZAC gene (human homolog of Zac1) and hormonal factors in the pituitary gland. In this way, many Zac1-expressing projections in the arcuate nucleus and eminence media are colocalized with some release hormonal factors, such as LHRH and CFR, during postnatal development (Fig. 4L–O; Table 2). All together, these findings suggest that Zac1 is implicated in the maturation of the neuronal endocrine hypothalamic system.

Postnatal Zac1-glutamatergic cells.

During postnatal developmental stages, the Zac1-expressing cells are not colocalized with glutamatergic receptors, except for a few cells in the deeper developmental cortical layer that are transitorily immunoreacted for GluR2/3. However, in adult mice, very few Zac1-expressing cells are colocalized with GluR2/3 receptors in the hippocampal formation (Table 2). Additionally, Zac1 is up-regulated in the granular layer of the dentate gyrus of the hippocampus after seizures induced by KA (Valente et al., 2004). These granular cells are essentially glutamatergic, although recent studies also show the presence of GAD67 in some granular cells (Schwarzer and Sperk, 1995; Sloviter et al., 1996). Thus, some glutamatergic cell subpopulations of the hippocampal formation express Zac1 during development and after injury, suggesting the involvement of Zac1 in the early plasticity processes related with excitatory neurons.

Taken together, these developmental results suggest that, in the CNS, there are several subpopulations of neural progenitor cells that express Zac1. Thus, in the lateral ventricles, Zac1 is expressed in the progenitor/stem cells of the ventricular and subventricular zones. However, if the new neural cells leave the ventricular zone and migrate to the cortical layers, the expression of Zac1 gene in these cells is completely down-regulated, whereas if the new neural cells follow the RMS, the expression of Zac1 is weakly down-regulated and persists in all of the olfactory system, even in adult stages. Moreover, Zac1 regulates the expression of the PACAP type 1 receptor gene (Hoffmann et al., 1998), and PACAP is implicated in the regulation of the development of the neuronal and glial precursors, because PACAP guides the transition from cell proliferation to cell cycle exit (for review, see Waschek, 2002). Therefore, the expression of Zac1 gene in the lateral neuroepithelia suggests an important coordination of the cell fate by Zac1 and PACAP, probably by the regulation of both proliferation and determination of the neural progenitor cells, as well as by the induction of the apoptotic process in restricted progenitor subpopulations. On the other hand, in the third and fourth ventricles, the expression of the Zac1 gene appears in the neural progenitor cells as in the neural cells (essentially GABAergic interneurons) that leave the subventricular zone and migrate toward their final position in the hypothalamic and brainstem area, respectively, where Zac1 gene is strongly expressed in the adult brain (Valente and Auladell, 2001). Therefore, Zac1 may play an important role in the differentiation of the several GABAergic subpopulatons.

Zac1 Expression in the Development of Vertebrate Skeleton

Positioning and patterning of the limb involves cellular interactions between the ectoderm surrounding the limb bud and the mesenchymal cells that form the core of the limb bud (Christ and Brand-Saberi, 2002). At E10–E12, Zac1 gene is expressed in the dorsal region of the neural tube (neural crest cells; Fig. 1B,K), in the notochord (Fig. 1K), in the somites (Fig. 1B,C), and in the apical ectodermal ridge of the limb bud (Table 1).

At E12.5, intense Zac1 gene expression is found within several cartilaginous sites of bones: in the craniofacial skeleton (such as Meckel's cartilage, tooth primordium, nasal cartilage, and maxillary bone), in the limb skeleton (such as radius, humerus, phalangeal, and metacarpal), and in the axial skeleton (such as ribs, body vertebrae, and thoracic vertebral body), as well as in many vascular cells of the limbs (Fig. 1C–E,H,L; Table 1). Immunohistological studies with Zac1 protein confirm the same expression pattern obtained with Zac1 gene (data not shown). At E14.5, intense expression of Zac1 gene is observed in cartilaginous and ossification bone sites (Fig. 1N–U; Table 1). However, at this embryonic stage, the immunolabeled for Zac1 protein is only detected in the chondrocytes and perichondrium cells, as well as in the vascular cells that surround the limb bones (Fig. 5A). At E16, the expression of Zac1 gene decreases strongly in the ossification sites and no detectable expression is found at E18, whereas the expression in chondrocytes is maintained throughout postnatal stages (Fig. 5B–F) and in adulthood. Therefore, the Zac1 gene is transitorily expressed in ossification bone sites, whereas its expression is permanent within cartilaginous sites.

Figure 5.

Immunolabeling for Zac1 in chondrogenic sites of the bone during development. A: At embryonic day (E) 14, in the ribs, Zac1 protein (in black) is found in high levels in the resting (CMMA, chondrocytes with moderate mitotic activity) and proliferating (CHMA, chondrocytes with high mitotic activity) chondrocytes and in low levels in the postmitotic chondrocytes (PMC). No detectable immunoreaction is found in bone (Bn). In addition, some Zac1-expressing cells are found in the perichondrium (solid arrows) as well as in the muscle cells (open arrowheads) that surround the cartilage anlage. B–G: At E16, Zac1 protein is found in all developing cartilaginous sites, especially in the limbs (B) and the ribs (D–G). All Zac1-expressing chondrocytes (in brown) in the CHMA are in proliferation, because they are colocalized with proliferating cell nuclear antigen (PCNA; in black; solid black arrows in D), whereas some of the Zac1-expressing chondrocytes in CMMA are not in proliferation (only labeled for Zac1, solid white arrows in D). E,F: Colocalization studies with Zac1 (in brown) and ssDNA-F7-26-Apostain (in black) show that some chondrocytes are double labeled in the postmitotic zone surrounding the bone site (solid white arrows in E), whereas no detectable colocalization is found in the CHMA and CMMA areas or in the perichondrium (solid black arrows in F) and muscle connective cells (arrowheads in F). G: The expression of Zac1 (in black) in chondrogenic sites is maintained in postnatal stages included the craniofacial cartilages. The sections in A–C and G are counterstained with methyl green–pyronin.

Three embryonic lineages are responsible in the formation and development of vertebrate skeleton: neural crest cells give rise to the craniofacial skeleton; paraxial mesoderm cells (somites) form the axial skeleton (vertebrae column and ribs), the dermis of dorsal skin, and the skeletal muscle of the body wall and the limbs; and lateral plate mesoderm cells form the limb skeleton (Erlebacher et al., 1995; Olsen et al., 2000). Chondrocytes arise from these three lineages (for review see Shum and Nuckolls, 2002), migrate toward specific embryonic locations where the skeleton will develop, form a dense nuclei of condensations, and then the chondrocytes or osteoblasts differentiate (Hall and Miyake, 2000; DeLise et al., 2000). Thus, at early embryonic stages, in the clavicles and in the majority of facial bones, the cells of mesenchymal condensations differentiate directly into osteoblasts (membranous bone development), whereas in the rest of bones (limb skeleton, ribs, vertebrae, and so on), the cells in mesenchymal condensations differentiate into chondrocytes to create a cartilaginous anlage of the future bone (endochondral skeletal development; for review, see Shum and Nuckolls, 2002; Horton, 2003). Chondrocytes show a life cycle of proliferation, differentiation, maturation, and apoptosis. In accordance with the present results, Zac1 is observed mainly in the endochondral skeletal development, in which they are detected in all cartilage primordia between E12 and E14, and where the expression of Zac1 in chondrocytes is differential, depending upon the cell cycle phase. Thus, four different classes of Zac1-chondrocytes are detected (Fig. 5A,E; Table 2): (1) the resting chondrocytes (quiescent) located within of the most remote region of the ossification zone, which express high levels of Zac1 and, consequently, show lower colocalization with PCNA or BrdU; (2) the proliferating chondrocytes located in the middle part of the chondrogenic site, which express high levels of Zac1, PCNA, and BrdU; (3) the differentiated chondrocytes in the most internal chondrogenic area (prehypertrophic chondrocytes), which express low-to-moderate levels of Zac1; and (4) the apoptotic hypertrophic chondrocytes located in the border of the ossification site, which express low levels of Zac1 and high levels of Apostain (Fig. 5E; Table 2). Therefore, as occurs in the embryonic nervous system, Zac1 expression is intense in proliferating chondrocytes and its expression is down-regulated when the progenitor cells leave the cell cycle.

In addition, hypertrophic chondrocytes induce sprouting angiogenesis from the perichondrium (Gerber et al., 1999). Vascular invasion from the perichondrium or bone collar brings osteoblast progenitors that will from ossification centers (Yang and Karsenty, 2002). Thus, with the formation of primary ossification centers, the cartilage matrix is degraded and the mature chondrocytes undergo apoptosis. Zac1 is expressed in the perichondrium and vascular cells that surround the limb bones sites, which are positively PCNA-labeled (Table 2), whereas their expression in the vascular interdigital zone overlaps with Apostain (Table 2).

Therefore, Zac1, like collagen type II, is expressed in all steps of the chondrogenesis (including mesenchymal condensations that occur between E10 and E12), which suggests a regulatory Zac1 role in the development of the skeleton, in which the proliferating chondrocytes differentiate into hypertrophic chondrocytes and facilitate the ossification spread with the degradation and replacement of the cartilage by apoptosis (Shum and Nuckolls, 2002). The present results suggest that Zac1 participates in all cellular steps of the chondrocyte life cycle, from proliferation to apoptosis, and they reinforce the previous reports of the expression of Zac1 gene in the primordium of the cartilage sites (Valente and Auladell, 2001; Tsuda et al., 2004) and of the ZAC gene (human homolog of Zac1) in adult bone marrow (Varrault et al., 1998).

Development of the Skeletal Muscle

At E12–E14, many migrating dermomyotomal cells of the limbs express the Zac1 gene (Fig. 1K,Q,R; Table 1), as well as some differentiated muscle cells surrounding the axial primordium cartilage skeleton (vertebrae column and ribs). In addition, many facial and tongue muscle cells express the Zac1 gene at E14 and during all embryonic stages. Many of these Zac1-expressing cells are positively labeled to Nestin, PCNA, BrdU, or Forse-1, which confirms their mitotic character, as well as to Netrin-1, which is implicated in the migratory process (Fig. 6; Table 2). At E16, the muscle Zac1-expressing cells are colocalized with Vimentin (intermediate filament proteins within cells of mesenchymal derivation; Table 2). Furthermore, at E15, it is possible to detect the Zac1 gene expression in the primordium of the nails. The epidermis is a derivate of the surface ectoderm that forms a protective barrier and specific appendages, including hair, nails, and different eccrine glands. The surface ectoderm also forms the epithelium of the oral cavity and tongue (Jonker et al., 2004). Moreover, Zac1 gene is expressed in the muscle cells that surround the follicle and in the primordium of follicle of vibrissae (Table 2), where some Zac1-expressing cells are colocalized with Nestin or PCNA. The follicle contains a distinct population of presumptive follicular stem cells that express Nestin (Amoh et al., 2004). Zac1 could be implicated in the regulation of mitotic activity of the stem cells of the follicle of vibrissae.

Figure 6.

Zac1 expression in muscle cells during embryonic development. A–C: Some Zac1-expressing skeletal muscle cells (in blue) are coexpressed with the progenitor/stem cell marker Nestin (in brown) in the tail (A), limbs (B), and craniofacial areas (C). D,E: Some Zac1-expressing skeletal muscle cells (in blue) are colocalized with the proliferating marker proliferating cell nuclear antigen (PCNA; solid arrows in D) and with migrating marker Netrin-1 (solid arrows in E), in the same areas described above. F: The immunolabeling for Zac1 protein is found in the same areas described for the Zac1 gene. G,H: As the development proceeds, the Zac1 expression is maintained in the skeletal muscle cells (in blue) and these cells colocalize with progenitor markers such as FORSE-1 (solid arrows). E, embryonic day.

All muscle cells produced by the somites take their origin from the dermomyotome located in the dorsal part of the somite, which receives signals from the notochord and the floor plate of the neural tube, initiating a de-epithelialization; the individual mesenchymal muscle precursor cells migrate, proliferate, and differentiate to form individual muscles (Christ and Ordahl, 1995; Schmidt et al., 1998; Borycki and Emerson, 2000). At E9–E10, the Zac1 gene is strongly expressed during somite formation (Piras et al., 2000; Valente and Auladell, 2001; Tsuda et al., 2004) and at E14–E18 in the migrating and proliferative mesenchymal muscle precursor cells (Fig. 6; Table 2), as well as in several waves of muscle fiber formation (myofibers) during development. However, in the postnatal stages and with the differentiating process of the muscle cells, expression of the Zac1 gene decreases and persists at low levels in some differentiated myofibers of the craniofacial, limb, and tail muscle cells. The present data reinforce the previous results obtained for ZAC and Zac1 genes in adult and fetal tissues (Varrault et al., 1998; Piras et al., 2000; Arima et al., 2000; Ma et al., 2004).

Therefore, during early embryonic stages, Zac1 gene is strongly expressed in progenitor cells of the skeleton and in the skeletal muscle tissues, as well as in apoptotic cells of the bone development. The ventral part of the somite (sclerotome) gives rise to the cartilage and bone of the vertebral column and ribs, whereas the dorsal part of somite (dermomyotome) produces the dermis of the back and the skeletal muscle of the body and limbs and the muscular connective tissue, except in the head muscles, which proceed from prechordal and paraxial mesoderm (Buckingham et al., 2003). The wide expression of the Zac1 gene in most of these tissues, as well as in most, if not in all, cellular phases, suggests that Zac1 could be a regulating control factor, like other transcription factors (Pax, bHLH, Sox, and so on) in chondrogenesis and myogenesis during embryonic development (Stockdale et al., 2000; Olsen et al., 2000; Moran et al., 2002; Yang and Karsenty, 2002).



We used embryo, postnatal, and adult NMRI (Iffa Credo, Lyon, France) mice. The day on which a vaginal plug was detected was considered E0. E1 began 24 hr later. The day of birth was designated P0. The fetal animals were removed from the mother under anesthesia by intraperitoneal injection of ketamine (100 mg/kg) and Xylazine (10 mg/kg). All animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer and processed for in situ hybridization (ISH) and immunohistochemistry (IHC). Alternatively, six NMRI pregnant mice were injected i.p. at E14, E16, and E18 (two mice for each age) with 50 mg/kg of BrdU (Sigma, in Tris-buffered saline, pH 7.6). Two hours after injection, the mice were killed and the embryos were collected, perfused, and fixed in 4% paraformaldehyde overnight. The animals were kept under controlled temperature, humidity, and light conditions, and they were treated according to European Community Council Directive 86/609/EEC and the procedure was registered at the Departament d'Agricultura, Ramaderia i Pesca of the Generalitat de Catalunya. Every effort was made to minimize animal suffering.


Antisense and sense riboprobes were labeled with digoxigenin-d-UTP (Boehringer-Mannheim). ISH was performed on free-floating tissue sections as described by Valente and Auladell (2001). Briefly, sections were pretreated with H2O2 and HCl and hybridized overnight at 61°C with antisense or sense digoxigenin–d-UTP-labeled riboprobes (mouse Zac1 cDNA used in Valente and Auladell, 2001; rat NPY cDNA; rat Nestin cDNA; mouse GAD65 cDNA; mouse EGFR1 cDNA). After ISH, sections were stringently washed in 50% formamide solutions at 61°C and incubated with 100 μg/ml RNase A (at 37°C). After that, the sections were blocked with 10% normal goat serum (NGS) and incubated overnight at 4°C with an alkaline phosphatase-labeled antidigoxigenin antibody (1:2,000; Boehringer-Mannheim). To view alkaline phosphatase activity, sections were incubated with nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate toluidine salt (BCIP). The anatomic analysis of the embryonic areas was made in accordance with “The Atlas of Mouse Development” by Kaufman (1999).

Double ISH-IHC Procedure

After obtaining an intense labeling for Zac1, Nestin, EGFR1, NPY, or GAD65 transcripts in the ISH, free-floating sections were incubated overnight at 4°C with one of the following primary antibodies: rabbit anti-Zac1 (1:1,000, L. Journot), anti-TH (1:2,000, Chemicon), anti-GFAP (1:2,000, Dako), anti-Ng2 (1:1,000, W.B. Stallcup), anti-calbindin (1:4,000, Swant), anti-calretinin (1:2,500, Swant), anti-parvalbumin (1:5,000, Swant), anti-LHRH (1:5,500, Chemicon), anti-CRF (1:3,500, Chemicon), anti-GluR1 (1:1,000, Chemicon), anti-GluR2/3 (1:1,000, Chemicon), anti-GluR4 (1:1,000, Chemicon), anti-GluR5/6 (1:800, Chemicon), anti-GAD65 (1:700, Chemicon), anti-somatostatin (1:1,000, Dakkopats), and mouse anti-PCNA (1:700, Chemicon), anti–Nestin-Rat-401 (1:200, hybridoma bank), anti-FORSE-1 (1:500, hybridoma bank), anti-BrdU (1:300, Roche), anti–β-tubulin (1:100, Chemicon), anti–Netrin-1 (1:300, Oncogene), anti–PSA-NCAM (1:7,500, G. Rougon), anti-vimentin (1:400, Dako), anti-NeuN (1:300, Chemicon), and mouse anti-ssDNA (F7-26) Apostain (1:150, Alexis). After that, sections were sequentially incubated with biotinylated goat anti-rabbit or horse anti-mouse antibodies (1:200), and then with the avidin-biotin-peroxidase complex (ABC, 1:200). Peroxidase was developed with 0.05% diaminobenzidine (DAB) and 0.01% H2O2.

IHC Techniques

IHC was performed on free-floating tissue sections as described by Valente et al. (2004). Briefly, free-floating sections were treated with 0.5% H2O2, blocked with 10% of NGS and incubated overnight with the Zac1 antibody and processed as described above until developed with 0.05% DAB–0.01% H2O2, for single immunohistochemistry, or with 0.05% DAB–0.01% H2O2–0.2% NiNH4SO4, for double immunohistochemistry. In this latter case, the immunolabeled sections were washed and incubated again with a second primary antibody and developed with 0.05% DAB–0.01% H2O2. Alternatively, some sections were counterstained with hematoxylin and methyl green–pyronin.

Detection of the In Situ DNA Fragmentation

Free-floating sections were treated following the protocol supplied by Klenow-FragEl DNA Fragmentation Detection Kit (Oncogene).


We thank Drs. Steven Sabol, Toshiyuki Takeuchi, Kunihiko Obata, and Harley Kornblum for providing us with NPY, Nestin, GAD65, and EGFR1 probes, respectively. We would also thank Drs. W.B. Stallcup, G. Rougon, and L. Journot for providing us with Ng2, PSA-NCAM, and Zac1 antibodies, respectively. We thank Tom Yohannan and Jordi Correas for editorial and technical assistance, respectively.