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Cell Growth & Differentiation Vol. 12, 351-361, July 2001
© 2001 American Association for Cancer Research

Expression of Ca2+/Calmodulin-dependent Protein Kinase IV (CaMKIV) Messenger RNA during Murine Embryogenesis1

Stephen L. Wang, Thomas J. Ribar and Anthony R. Means2

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric, multifunctional serine/threonine protein kinase that is expressed in subanatomic regions of the central and peripheral nervous system, T lymphocytes, and male germ cells. It is frequently localized to the nucleus, where it serves as a mediator of Ca2+-dependent gene expression. Although CaMKIV expression in the adult rat central nervous system and thymus has been described, little is known about the embryonic expression of murine CaMKIV. Here we report a thorough embryonic expression study of CaMKIV mRNA from embryonic day 9.5 through postnatal day 1. Expression patterns during embryonic development are significantly different from those of adults, suggesting specific roles for CaMKIV during development. Regions of high CaMKIV mRNA expression include thymic and bone cartilage primordia as well as specific cranial nerve ganglia (trigeminal, vestibulocochlear, and glossopharyngeal), thalamus, and dorsal root ganglia. This pattern of expression is chronologically consistent with periods of extensive cellular differentiation, proliferation, or neuronal survival selection and shows a predilection for neural crest-derived cells. These trends, along with recent studies in the CaMKIV null mouse, suggest that CaMKIV may play an important physiological role in cellular differentiation during embryogenesis.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
CaMKIV3 is a member of the broad substrate specificity class of CaM kinases that also includes CaMKI, CaMKII, and the CaM kinase kinases (1, 2, 3, 4) . Until recently, CaMKIV expression was thought to be limited to subanatomical regions of the central and peripheral nervous system (5, 6, 7) , T lymphocytes, and male germ cells (8 , 9) . Although CaMKIV is found to some extent in the cytoplasm, it is more conspicuous in the nucleus of cells in which it is expressed (10) . In the nucleus, CaMKIV is a component of a signaling complex that includes the protein phosphatase PP2A and has been implicated in the regulation of transcription (11) . However, although recent evidence suggests that CaMKIV may function as a CREB kinase in vivo (12 , 13) , the physiologically relevant substrates for CaMKIV remain to be identified conclusively.

We hypothesized that analysis of the phenotypic consequences caused by the absence of CaMKIV in mice would help lead to identification of the physiologically important targets of this enzyme in the adult. The results of this effort to date have led to some surprising physiological findings. Adult males are infertile because of specific impairment of spermatogenesis in late elongating spermatids (14) . Here the sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine 2 and retention of transition protein 2 in step 15 spermatids. These data provide the first description of a nuclear function for CaMKIV that is not related to gene transcription, and they emphasize a role for CaMKIV in the terminal differentiation of male germ cells. These results in testis were the forerunner to our current hypothesis that CaMKIV is involved in differentiation of cells in which it is expressed. Indeed, CaMKIV is expressed in ovarian granulosa cells, and it plays a role in the regulation of female fertility (15) . The Camk4-/- female mice show profoundly impaired fertility, which seems to be because of defective ovulation secondary to abrogation of granulosa cell differentiation. In the cerebellum of young adult mice, there is a deficit in the number of Purkinje cells and in the maturation of the remaining Purkinje cells that normally occurs during the second week of postnatal life (13) .

As illuminating as these adult requirements for CaMKIV have proven to be, we were surprised to find that only 50% of the mice genotyped at weaning were nullizygous (14) . The remaining 50% of the Camk4-/- mice, in whom the deletion was maintained on a mixed 129/SV x C57BL/6J genetic background, appeared to die late in gestation or immediately after birth. We have bred the gene deletion onto 129/SV and C57BL/6J genetic backgrounds but have yet to produce a single live null mouse. We suspect that this deletion results in fully penetrant embryonic lethality in these two genetic strains, and we are in the process of investigating the cause of death in these mice. However, nothing has been reported in the literature regarding the expression of CaMKIV during mouse embryonic development. As a prelude to identifying the CaMKIV targets that are essential for mouse development, we have performed an analysis of Camk4 gene expression during embryogenesis by in situ hybridization. These experiments yielded several unexpected results in both the timing and distribution of expression.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Tables 1Citation and 2Citation summarize the data for the developmental expression of CaMKIV mRNA in the mouse from embryonic day E9.5 through postnatal day P1. As described in "Materials and Methods," serial sections were used for sense and antisense riboprobes, and the results obtained were consistent for three probes derived from different regions of the CaMKIV cDNA. The rating scale used to score the relative intensity of hybridization signal presented in Tables 1Citation and 2Citation was necessarily subjective. From initial evaluation of the sections hybridized with all three of the antisense probes, the most intense signal appeared to be the cranial nerve expression at day E15.5. Therefore, this signal was assigned a numerical value of 5. In each experiment, the numerical ratings are indicative of the relative difference in signal obtained between antisense and sense probes applied to serial sections of an embryo, and serial sections of day E15.5 hybridized to the same antisense and sense probes were included to indicate the maximal signal:noise ratio (5) . This process helped to minimize the differences in background that occurred from experiment to experiment because of differences between radiolabeled probe preparations. In both embryonic and adult rats, CaMKIV mRNA and protein expression patterns have been demonstrated both temporally and spatially in peripheral tissues such as the thymus as well as specific neural regions such as the cerebellum, hippocampus, cortical neurons, thalamus, and some peripheral ganglia (5, 6, 7, 8, 9) . Our results based on analysis of in situ hybridizations performed on mice from day E9.5 to day P1 confirmed the expression of CaMKIV in these tissues (Tables 1Citation and 2Citation ). It should be noted however, that although most the patterns of expression such as those seen in the hippocampus could be overlapped with the rat, we also observed the presence of CaMKIV mRNA in several other developmentally age-specific tissues and organ systems not reported previously to express CaMKIV in rats (Tables 1Citation and 2Citation ).


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Table 1 Summary of early developmental CaMKIV expression

 

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Table 2 Summary of later developmental CaMKIV expression

 
Developmental Expression of CaMKIV in Peripheral Tissues
Nonneuronal Expression Patterns.
As shown in Fig. 1ACitation , the first branchial arch is a structure that is clearly discernable in early murine embryos. As shown in Fig. 1BCitation , we detected the earliest high expression of CaMKIV mRNA in murine embryogenesis at day E9.5 in the first branchial arch, and the hybridization signal remained strong through day E11.5 (Table 1)Citation . Transcripts were concentrated in the epithelium of the maxillary and mandibular processes of the first branchial arch. Additionally, low expression levels of RNA transcripts in the derivatives of the first arch were also detected throughout development (Tables 1Citation and 2Citation ).



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Fig. 1. CaMKIV mRNA embryonic expression as detected in the epithelium of the maxillary and mandibular components of the first branchial arch at day E9.5 and CaMKIV mRNA expression in the developing incisor tooth primordia of day E15.5 mouse embryos. A, H&E-stained sagittal section of day E9.5 mouse at x50, detailing first branchial arch. B, day E9.5 sagittal mouse section hybridized with CaMKIV antisense probe 3 (x50). C, H&E-stained sagittal section of primordial incisor tooth detailing normal anatomy (x100). B, x100 darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 3 showing specific CaMKIV mRNA expression in odontoblasts. 1B, first branchial arch; TP, incisor tooth primordia.

 
Beginning at day E10.5, CaMKIV mRNA transcripts were detected in the presumptive regions of first molar tooth germ and in the thickened dental epithelium. By day E13.5 the bud, cap, and bell stages of the lower incisor tooth primordia developed and could be seen clearly in the embryo throughout the remaining development (Fig. 1C)Citation . Strong expression of CaMKIV mRNA was detected in the developing lower incisor from day E13.5 through day P1 (Tables 1Citation and 2Citation ), with transcripts primarily being expressed by neural crest-derived odontoblasts (Fig. 1D)Citation .

By day E12.5, the bone primordia begin to enter a period of development during which the dynamics of cellular differentiation, proliferation, and ossification of bone are being established (16) . At this period of development, skeletal structures became more clearly defined (Fig. 2, A and C)Citation , and moderate to strong CaMKIV mRNA expression corresponding to nonossified cartilage primordium regions was detected. The earliest transcripts were evident in vertebral primordia beginning at day E12.5 and persisting through day P1 (Tables 1Citation and 2Citation ). Transcripts were also detected in the midshaft rib cartilage primordia from day E14.5 onward, whereas expression in the iliac bone cartilage primordia was seen from E15.5 onward (Fig. 2, B and D)Citation . Moderate levels of CaMKIV mRNA expression were observed in all of the bone-related primordia, but they peaked in the E15.5 sections.



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Fig. 2. CaMKIV mRNA expression in the nonossified cartilage primordial regions of the ribs and iliac bones at day E15.5. A, H&E-stained sagittal section of cartilage primordia of the ribs detailing normal anatomy (x50). B, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 3 showing expression in the cartilage primordia (x50). C, H&E-stained sagittal section of cartilage primordia and ossification center of the developing iliac bone (x50). D, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 3 showing CaMKIV mRNA transcripts in the cartilage primordial regions (x50). RP, cartilage primordia of ribs; I, cartilage primordia of iliac bone.

 
Glandular Expression.
By day E12.5, the thymic primordium also becomes a very pronounced structure as it undergoes extensive development (Fig. 3A)Citation . At this stage of development, the thymic primordium is not yet vascularized, and stem cells leaving adjacent vessels must traverse the peripheral mesenchyme and basement membrane to enter the primordium (17) . These cells in the periphery may represent uncommitted hematopoietic cells and early T-cell precursors crucial to future thymic development (18) . Our in situ hybridization results indicated that by day E12.5, CaMKIV mRNA was expressed strongly in the thymus primordium with the localization of mRNA transcripts primarily in the periphery (Fig. 3B)Citation . From day E13.5 onward, the localization pattern changed and became diffuse, and the CaMKIV hybridization signals were no longer restricted to the periphery of the organ but were found to reside within the thymocytes (data not shown). CaMKIV mRNA expression persisted at high levels throughout the remaining development of the thymus, and the signal was observed with a similar intensity in sections from P1 pups (Table 2)Citation .



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Fig. 3. CaMKIV mRNA expression in the day E12.5 thymic primordium and in the submandibular gland of E15.5 mouse embryos. A, H&E-stained x100 of thymic primordium sagittal section. B, darkfield view of thymic primordium hybridized with CaMKIV antisense probe 2 (x100). Note that the CaMKIV mRNA transcripts are localized to the periphery of the thymic primordium. C, H&E-stained sagittal section of submandibular gland at x50 detailing normal anatomy. D, x50 darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 1 showing specific localization of CaMKIV mRNA transcripts in immature acini cells and developing excretory ductal cells. Th, thymic primordium; SG, submandibular gland.

 
Expression of CaMKIV mRNA was also detected in the developing submandibular gland (Fig. 3C)Citation . Beginning at day E14.5, weak expression could be detected in the gland (Table 1)Citation . By day E15.5, weak signal could be detected in the immature acini cells, whereas intense expression was seen in the developing excretory ductal cells (Fig. 3D)Citation . Transcripts remained expressed at high levels through day P1 (Table 2)Citation . Additionally, CaMKIV mRNA was detected in the developing adrenal gland at E16.5 and E18.5, where diffuse expression was seen in both the cortical and medullary regions (Table 2)Citation .

Nervous System Development and CaMKIV Expression
Cranial Nerves and Related Structures.
Time-dependent expression of CaMKIV mRNA in the cranial nerves and related structures during development was observed. Strong expression of CaMKIV mRNA was first noted at day E10.5 in the optic stalk (Fig. 4)Citation , the precursor of the optic nerve which begins, along with the optic cup, as an evagination of the developing diencephalon (19 , 20) . Optic stalk expression persisted at day E11.5, and a similar level of expression was seen in the optic tract at day E12.5 (Table 1)Citation ; however, no expression was observed later in the developing optic nerve. Strong mRNA expression was also seen in the olfactory epithelium beginning at day E11.5 (Table 1)Citation . Expression remained at high levels throughout development (Fig. 5E)Citation and was still clearly visible in day P1 sections. However, no expression was observed in the developing olfactory bulbs.



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Fig. 4. CaMKIV mRNA expression in the day E10.5 optic stalk. A, day E10.5 sagittal mouse section hybridized with CaMKIV sense probe 3 (x50). B, E10.5 sagittal mouse section hybridized with CaMKIV antisense probe 3 (x50) showing CaMKIV mRNA expression in the optic stalk. OS, optic stalk; LOS, lumen of optic stalk.

 


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Fig. 5. CaMKIV mRNA expression in both the day E12.5 and day E15.5 mouse embryo. Serial sagittal sections are all at x12.5 (A–C and D–F). A, H&E-stained sagittal section detailing anatomical locations of interest at x12.5. B, sagittal mouse section hybridized with CaMKIV antisense probe 2, indicating expression in the pontine flexure, vestibulocochlear ganglion, trigeminal ganglion, thymic rudiment, and marginal layer of spinal cord. C, control sagittal mouse section hybridized with CaMKIV sense probe. D, H&E-stained sagittal section detailing normal anatomy of CaMKIV expression areas. E, darkfield view of sagittal section hybridized with CaMKIV antisense probe 3 showing strong expression of mRNA transcripts in the thalamus, frontal cortex, pontine flexure, vestibulocochlear ganglion, trigeminal ganglion, glossopharyngeal ganglion, and olfactory epithelium. F, control darkfield view of sagittal section hybridized with CaMKIV sense probe. C, vestibulocochlear ganglion; Mn, mantle layer of spinal cord; Mr, marginal layer of spinal cord; PF, pontine flexure; Th, thymic primordium; V, trigeminal ganglion; F, frontal cortex; IX, glossopharyngeal ganglion; OE, olfactory epithelium; T, thalamus; V, trigeminal ganglion; V2, maxillary branch of trigeminal ganglion.

 
By day E11.5, CaMKIV transcripts began to appear throughout the developing cranial nerves. This expression correlates to a period of extensive development during which ganglion proliferation, innervation, and apoptosis proceed (21, 22, 23) . The trigeminal ganglion, which innervates several somatosensory type receptors for the face, oral cavity, and nasal cavity (21) , expressed transcripts that persisted at very high levels throughout development. The hybridization signal was diffuse in both the early, single aggregate of cells (Fig. 5B)Citation , as well as the late, bilobed ganglion structure (anteromedial and posterolateral lobes; Figs. 5ECitation and 6, A and BCitation ). Similarly, the facioacoustic ganglia expressed low to moderate levels of CaMKIV mRNA beginning at E11.5, when they were still contiguous, and expression continued through development at moderate to high levels in their derivatives (Tables 1Citation and 2Citation ). The facial ganglia were also observed to express high levels of CaMKIV mRNA at E15.5. The glossopharyngeal (IXG) and vagal ganglia (XG) also expressed CaMKIV mRNA from day E11.5 within the inferior IX/X contiguous area. At day E13.5, strong mRNA expression was also observed in the superior IX/X region, and by day E14.5, when the individual ganglia were distinguishable, the highest levels of expression were seen in the petrosal (inferior IXG) and nodose (inferior XG) ganglia (Figs. 5ECitation and 6, C and DCitation ). Finally, the vestibulocochlear ganglia, although first recognized around day E12, did not express CaMKIV mRNA until day E13.5. As a note, the independent ganglia of this nerve do not separate until around day E16 (24) , and correspondingly both individual cochlear and vestibular ganglia showed high expression at E15.5 and E16.5 (Figs. 5ECitation and 6, C and DCitation ).



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Fig. 6. Specific areas of CaMKIV mRNA expression at day E14.5 (G and H), day E15.5 (A–F, I, and J), and day E16.5 (K and L) from serial sagittal sections of mouse embryos at x50. A, H&E-stained sagittal section detailing normal embryonic mouse anatomy of the bilobed trigeminal ganglion. B, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 3 showing diffuse, strong expression of CaMKIV mRNA transcripts in the anteromedial and posterolateral lobes of the trigeminal ganglion. C, H&E-stained section detailing normal anatomy of E15.5 vestibulocochlear and glossopharyngeal ganglia. D, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 3 showing strong, diffuse expression of CaMKIV mRNA transcripts in the vestibular, cochlear, and glossopharyngeal ganglia. E, H&E-stained section detailing normal anatomy of E15.5 dorsal root ganglia. F, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 2 showing diffuse mRNA expression in the dorsal root ganglia. G, H&E-stained section detailing normal anatomy of E14.5 frontal cortex. H, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 2 showing CaMKIV mRNA expression in the frontal cortex (primarily in the intermediate zone). I, H&E-stained section detailing normal anatomy of E15.5 dorsal thalamus and parietal cortex. J, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 3, showing expression in the dorsal medial thalamus and parietal cortex. K, H&E-stained section detailing normal anatomy of E16.5 hippocampus and centromedial thalamus. L, darkfield view of serial sagittal section hybridized with CaMKIV antisense probe 1 showing CaMKIV mRNA transcripts localized to the hippocampus (primarily CA3 region and dentate gyrus) and centromedial thalamus. cmt, centromedial thalamus; D, dorsal root ganglia; dmt, dorsomedial thalamus; F, frontal cortex; H, hippocampus; IX, glossopharyngeal ganglion; P, parietal cortex; V, trigeminal ganglion; VIII, vestibulocochlear ganglion; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone.

 
Spinal Cord and Regional Brain Expression.
In the developing spinal cord, very low expression was detected in the mantle layer as early as day E9.5 (Table 1)Citation . Increased expression was not observed until day E12.5, when the level of CaMKIV mRNA in the marginal layer became more intense than that in the mantle layer and persisted through day 16.5 (Fig. 5B)Citation . By day E18.5, the hybridization signal reversed and became more intense in the mantle layer than the marginal layer (Table 2)Citation . CaMKIV mRNA transcripts were also detected in the developing dorsal root (Fig. 6, E and F)Citation and sympathetic ganglia. Weak dorsal root ganglia expression was first seen at day E11.5, and by day E12.5 onward, strong expression was noted in dorsal root ganglia from all spinal segments. High transcript levels were also noted in the sympathetic ganglia (cervical and celiac ganglia) at day E16.5.

Throughout both cortical and noncortical regions of the developing embryonic brain, moderate to strong CaMKIV mRNA expression was detected beginning from day E12.5 onward (Tables 1Citation and 2Citation ). Expression of CaMKIV mRNA in both the frontal and parietal cortical regions was first observed at day E12.5 and continued to be detected through day P1. By day E14.5, the highest levels of expression in the cortices were seen in the intermediate zone when compared with the ventricular zone, and very little expression was seen in the cortical plate (Fig. 6, G and H)Citation .

Other specific regions of high CaMKIV mRNA expression that became apparent by day E12.5 included midbrain structures such as the pontine flexure (Fig. 5, B and E)Citation . Although not shown clearly, the locus coeruleus nuclear complex, the pontine nuclei, raphe nuclear complex, and red nuclei also expressed CaMKIV transcripts (Table 2)Citation . Strong expression of CaMKIV mRNA was also detected in the dorsal thalamus (Fig. 5E)Citation . Although weak expression was seen as early as day E11.5, high expression in this structure was first observed at day E13.5 (Tables 1Citation and 2Citation ), which coincides with a crucial period of significant interaction between the brainstem and thalamus that results in extensive axonal migration and neuronal differentiation (25 , 26) . By day E15.5 and onward, a strong hybridization signal could be detected in all thalamic nuclei (Fig. 6, J and L)Citation . Other regions that had notable expression levels included the nucleus raphe subregions, the medulla oblongata, and the cuneate nucleus (Table 2)Citation .

Other neurological areas of strong CaMKIV mRNA expression included the hypothalamus and the hippocampus. Hypothalamic expression was observed as early as day E13.5, and it remained throughout prenatal development. Specific regions of moderate expression at day E16.5 included the zona incerta, the mammillary nuclei, and the anterior and the supraoptic nuclei (Table 2)Citation . Transcripts for CaMKIV in the hippocampus were also first noted at day E13.5, and they persisted to day P1 (Table 2)Citation , with the highest intensity signals in pyramidal neurons of the CA3 region and the dentate gyrus (Fig. 6 L)Citation . Perhaps the most surprising result in the neural tissues was the absence of CaMKIV mRNA expression in the embryonic cerebellum. Unlike CaMKIV expression in the embryonic rat Purkinje cells (5 , 6 , 27 , 28) , little to no hybridization signals were observed in any cerebellar cells during embryogenesis (Table 2)Citation . CaMKIV mRNA was first detected at low levels in mouse at day P1 within the early developmental stages of the internal granule cell layer. No mRNA transcripts were detected in the external granule cell layer or the molecular layer. Transcripts also appeared in the Purkinje neurons by day P3 (data not shown).


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Our findings reveal that the developmental expression of murine CaMKIV mRNA is not the same as in the adult, and that multiple tissues, not described previously, exhibit embryonic expression of CaMKIV mRNA. The strongest hybridization signals were found in the developing thymus, thalamus, and sensory neurons of the central nervous system and peripheral nervous system. Developmentally, much of the CaMKIV mRNA expression is in tissues derived from the neural crest, including the dorsal root ganglia, facioacoustic/vestibulocochlear ganglia, glossopharyngeal ganglia, glossopharyngeal-vagal ganglia, trigeminal ganglia, and odontoblasts of tooth primordia. The expression periods in these tissues correlate well with periods of significant differentiation, terminal mitoses, and cell survival selection, suggesting that CaMKIV may play an integral part in embryonic cellular differentiation.

The first significant expression of CaMKIV mRNA was seen in the first branchial arch at day E9.5, which could be related to axonal migration from the developing trigeminal ganglion. From day E11.5 onward, the trigeminal ganglion exhibited the strongest CaMKIV mRNA expression intensity among all other tissues. Prior immunohistochemical studies in embryonic rats showed that immunoreactivity first appeared in the trigeminal ganglion at day E15 with full expression at day E18 (6) . Interestingly, in our study several of the target tissues innervated by the trigeminal ganglion expressed CaMKIV mRNA during development, notably the maxillary and mandibular processes/epithelia, tongue, primordial teeth, and the first branchial arch. During normal development, the trigeminal ganglion becomes morphologically discernible on day E9, and the pioneer nerve fibers emerge from the ganglion and grow toward the periphery by day E9.5 (21 , 22) . From day E8.5 to day E12, a significant portion of trigeminal neuronal differentiation occurs (22) . Secondary fibers begin to appear by E11, and satellite cells appear in the trigeminal ganglion by day E15.5 (23) . The earliest fibers reach their peripheral targets by day E10.5, and the last cohort of neurons generally reach their targets by day E15 (21) . Finally, from days E12 to E19, a majority of the neurons in the ganglia degenerate and die (21) . Thus, the developmental expression of CaMKIV mRNA in the trigeminal ganglia and its target tissues parallels periods of extensive neurotrophic growth, neuronal differentiation, and survival selection/apoptosis.

This pattern of trigeminal and trigeminal target tissue expression of CaMKIV mRNA may relate to neurotrophin regulation. Neurotrophin theory asserts that developing neuronal survival depends upon the supply of one or more neurotrophins that are synthesized by their target fields (29) . Many of the developing sensory ganglia (trigeminal, dorsal root, nodose, and vestibulocochlear) are responsive to multiple neurotrophins including BDNF, nerve growth factor, and neurotrophin 3 (30, 31, 32) . These factors are crucial in the development and maintenance of the nervous system, regulating such basic processes as neuronal survival selection, neuronal differentiation, and target-derived neural signaling. It has been proposed that one of these neurotrophins, BDNF, is regulated by CaMKIV. Shieh et al. (33) have suggested that CaMKIV may regulate BDNF gene expression in cortical neurons, whereas Finkbeiner et al. (34) have presented evidence that CaMKIV is involved in the pathway by which BDNF controls CREB phosphorylation in its target cells. However, although our expression data are consistent with these results, the implications of CaMKIV-mediated BDNF pathways in embryonic development remain largely unexplored.

Our study also shows strong CaMKIV mRNA expression in the vestibular and cochlear cranial nerve ganglia and their structural predecessors, the vestibulocochlear ganglia and facioacoustic ganglia. At day E12, cranial nerves VII and VIII are not distinct, and they comprise the statoacoustic ganglion. But by day E13, this ganglion undergoes internal differentiation such that the portions of the ganglion that will give rise to the vestibular and cochlear ganglions become discernable (35) . Terminal mitosis of the vestibulocochlear ganglion occurs at day E12, the same day that the majority of the vestibular ganglion forms, whereas the cochlear ganglion forms on days E13-E14 (36) , and differentiation of hair cells begins at day E14 (37) . After this terminal mitosis, apoptosis peaks approximately 2–3 days later at day E13.5 in the vestibular ganglion and at days E15.5-E16.5 in the cochlear ganglion. Once again, the chronological order of CaMKIV embryonic expression in the vestibulocochlear ganglion found in our study correlates well with the developmental requirement of vestibular neurons for BDNF described by Bianchi et al. (38) .

Interestingly, CaMKIV can mediate the Ca2+-dependent transcriptional activation of immediate early genes of the fos and jun families in a number of cells and tissues (39) , and both have been implicated in inner ear development (40) . Studies in chick embryos have detected c-fos in the vestibulocochlear ganglion, expressed in a developmentally regulated pattern with the highest levels occurring before the highest rate of cell proliferation (41) . We have shown that CaMKIV mRNA is expressed in the vestibulocochlear ganglia during these periods. Therefore, given the putative regulatory relationship between CaMKIV and BDNF, it is provocative to speculate that CaMKIV may mediate embryonic BDNF expression and thereby regulate the induction of c-fos in embryogenesis or that BDNF may be mediating embryonic CaMKIV regulation of c-fos.

In addition to its nervous system functions, CaMKIV is known to play important roles during T-cell development. Anderson et al. (42) showed that transgenic mice specifically expressing a catalytically inactive form of CaMKIV in thymocytes demonstrated markedly decreased thymic cellularity with the remaining thymocytes showing a substantial decrease in IL-2 production secondary to abrogation of CREB phosphorylation. More recent studies in mice nullizygous for CaMKIV indicate that CaMKIV is important for the activation of CD4+ memory T cells.4 In the memory cells, CREB phosphorylation is markedly reduced, CREB-dependent immediate early genes are compromised, and cytokine genes such as IL-2, IL-4, and IFN-{gamma} are not transcribed. Our data show that CaMKIV mRNA can be detected at day E12.5, and intense expression persists through day E16.5, at which time the hybridization signal seems to diminish slightly in intensity. Interestingly, IL-4 production during murine embryonic development is known to peak at two time points, day E14 and day E16 (43) , raising the possibility that CaMKIV modulates IL-4 transcription and may thereby influence critical stages of T-cell development/differentiation during embryonic development.

One of the surprises of our study was the finding that CaMKIV mRNA is expressed in bone primordia beginning at E12.5. The appearance of primary ossification centers within the bones varies by the bone involved, but for most of the long bones, including the iliac and ribs, ossification centers are first seen from days E14.5 to 16.5 (16 , 19) . Concomitantly, differentiation of early osteoblastic cells occurs, and osteoclast progenitor differentiation and proliferation commence at this time (44 , 45) . Although the timing of this differentiation period in the bone primordia and CaMKIV expression may be coincidental, recent experiments in our laboratory indicate that mice nullizygous for the Camk4 gene have profound defects in long bone ossification.5 Given the differentiation defects seen in multiple cell types in Camk4-/- mice, it may be possible that CaMKIV mediates the expression of ossification factors or other cytokines.

Despite the diversity of tissues and cells expressing CaMKIV throughout embryonic development, a few notable trends emerge from this study: (a) there is extensive expression of CaMKIV mRNA in the developing nervous system with the highest levels of expression seen in sensory neural ganglia, including several cranial nerve ganglia and their target tissues; (b) CaMKIV mRNA is expressed early in hematopoietic-related tissues including the developing embryonic thymus and specific areas of developing bone cartilage primordia; and (c) high levels of CaMKIV mRNA were observed in tissues that are colonized by neural crest cells (dorsal root ganglion, facioacoustic/vestibulocochlear ganglion, glossopharyngeal ganglion, glossopharyngeal-vagal ganglion, trigeminal ganglion, and odontoblasts of tooth primordia). Moreover, these developmental patterns coincide temporally with periods of significant cellular differentiation, axonal migration in the nervous system, and neuronal survival selection. These correlations with differentiation are also evident in adult CaMKIV null mice. Our studies have revealed terminal differentiation defects in several cell types: CD4+ memory T cells fail to activate;4 postnatal maturation of Purkinje cells is stalled (13) ; ovulation is compromised (15) ; and specific steps in the terminal differentiation of spermatozoa fail (14) . The results from this study suggest that CaMKIV may play pivotal roles in cellular differentiation and development, and they provide a necessary embryonic basis for further investigation of the remarkable panoply of phenotypes found in the CaMKIV null mouse.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In Situ Hybridization of Staged Murine Embryos.
Three cDNA templates for riboprobes were generated by PCR from the CaMKIV mouse cDNA. These PCR fragments were subcloned into PCR®2.1 (Invitrogen Corp., Carlsbad, CA) plasmid vectors in both orientations to serve as template for transcription of antisense and sense riboprobes.

For probe 1, a 322-nucleotide fragment from the 3' translated region of exon XII (cDNA sequence 1112–1433) was generated from a full-length (1.4-kb) CaMKIV cDNA in a PCR®2.1 (Invitrogen) plasmid vector by PCR with the following primers: forward primer, 5'-CTTCAACCCAAGATGCCAAGG-3'; and backward primer, 5'-TCCTGCTGTGGAACCCCAAGTC-3'.

For probe 2, a 357-nucleotide fragment from the 3' and untranslated regions (204 nucleotides untranslated) of exon XII (cDNA sequence 1251–1607) was generated from cDNA sequence range 1251–1795 in a pGEM-based plasmid vector by PCR with the following primers: forward primer, 5'-TGCAGGTGTAAAAGAGGAGGAGAC-3'; and backward primer, 5'-GCACTGACATTAGGGTTACCAACTG-3'.

For probe 3, a 315-nucleotide fragment from exons VI/IX (cDNA sequence 420–735) was generated from mouse testis RNA by reverse transcription-PCR with the following primers: forward primer, 5'-GATTGTGGAGAAGGGATACTAC-3'; and backward primer, 5'-AGGATGTAGGTGATTATTCCT-3'.

These cDNA templates were linearized with appropriate restriction enzymes and transcribed in vitro using T7 polymerase in the presence of 35S-labeled UTP (Amersham, Piscataway, NJ) to generate sense and antisense riboprobes. All embryo sections as well as laboratory space to conduct the experiments were provided as a generous gift from Dr. Jeffrey Leiden (University of Chicago, Chicago, IL). Embryo collection, sectioning, and in situ hybridization were performed as described previously (46 , 47) using CD1 mouse embryos collected from timed pregnant females. Mice were housed at the University of Chicago under a 12-h light/12-h dark cycle with continuous access to food and water. The results obtained with each of the three antisense riboprobes were virtually identical, and each probe was hybridized to sections prepared from at least three embryos at every stage of development examined. Hybridizations were simultaneously performed with radiolabeled sense riboprobes to consecutive serial sections to detect nonspecific background. Slides were coated with photographic emulsion (Eastman Kodak, Rochester, NY) exposed for 7 days and developed following the manufacturer’s instructions (Eastman Kodak). Hybridization signals were visualized with epifluorescence and darkfield microscopy on a Zeiss Axioscope microscope (Carl Zeiss, Thornwood, NY). Separate serial sections were also stained with H&E for facilitating the identification of anatomical structures. Images were captured on color film (Kodak ASA 400) and transferred into Adobe Photoshop V5.0 (Adobe Corp., Mountain View, CA), where they were converted to non-color and assembled into figure panels.


    Acknowledgments
 
We are indebted to Dr. Jeffrey M. Leiden (now at Abbott Laboratories) for hosting S. L. W. in his laboratory at The University of Chicago and enabling this project to proceed, and we thank our colleagues Bill Wetsel and Ethan Corcoran, who provided invaluable help with the manuscript. We are also extremely grateful to Margaret Veselits (University of Chicago) for superb advice with in situ hybridization protocols and Fang Jiang (University of Chicago) for her expertise with embryo sectioning. Finally, we thank Hugh Crenshaw (Duke University, Durham, NC) for the use of microscopy equipment.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Howard Hughes Medical Institute medical student fellowship (to S. L. W.) and NIH Grant HD-07503 (to A. R. M.). Back

2 To whom requests for reprints should be addressed, at Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27710. Phone: (919) 681-6209; Fax: (919) 681-8461; E-mail: means001{at}mc.duke.edu Back

3 The abbreviations used are: CaMKIV, Ca2+/calmodulin-dependent protein kinase IV; CREB, cyclic AMP response element binding protein; BDNF, brain-derived neurotrophic factor; IL, interleukin. Back

4 K. A. Anderson and A. R. Means, unpublished observations. Back

5 C. M. Kitsos and A. R. Means, unpublished data. Back

Received for publication 2/19/01. Revision received 5/22/01. Accepted for publication 5/24/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
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